Stem cell inhibiting proteins

ABSTRACT

Proteinaceous molecules with stem cell inhibition activity are analogues of LD78 or MIP-1α which have mutations to prevent or reduce multimer formation beyond certain stages (for example a dodecamer). Aggregate formation is therefore inhibited, and the resulting low molecular weight monomers (or oligomers) have improved solution properties leading to enhanced productivity and greater therapeutic utility as stem cell protective agents, which are useful in tumour therapy.

This application is a division of application Ser. No. 07/982,759, filedas PCT/GB 92/02390 Dec. 23, 1992.

This invention relates to proteinaceous compounds having the propertiesof inhibitors of stem cell proliferation. In particular, the inventionrelates to engineered variants of protein molecules with stem cellinhibition activity, their preparation and pharmaceutical compositionscontaining them and their use as adjuncts to chemotherapy orradiotherapy, for example in the treatment of cancer.

The diverse cells of the haemopoietic system are derived frommultipotential stem cells by a process of sequential division anddifferentiation. The proliferation of the stem cell population iscontrolled in part by an inhibitory molecule produced by bone marrowmacrophages (Lord et al., Brit. J. Haematol. 34 441, (1976)). The murinehaemopoietic stem cell inhibitor has been shown to be an 8 kDa protein,macrophage inflammatory protein-1 alpha (MIP-1α (Graham etal., Nature344 442, (1990)). The properties of the stem cell inhibitor includeprotecting stem cells from the toxic effects of cell cycle specificcytotoxic agents (Lord and Wright, Blood Cells, 6 581 (1980)). Stem cellinhibitors therefore have enormous clinical potential as agents toprotect the stem cells from the chemotherapy or radiotherapy regimesused in tumour therapy. Additionally, stem cell inhibitors may be usedin the treatment of hyperproliferative diseases such as psoriasis,either alone or in conjunction with cytotoxic agents. Amino acidsequence homologies suggested that either the human LD78 or ACT2 geneproducts were the human homologues of the murine stem cell inhibitor(FIG. 1a) (Schall, Cytokine 3 165-183 (1991)). It has been demonstratedthat the human LD78 gene product is the functional homologue of murineMIP-1α, (Pragnell CRC Beatson Laboratory Scientific Report pp 21-25,(1990), Dunlop et al., Blood 79:2221-2225 (1992)).

As a component of the present invention, the secondary and tertiarystructure of LD78 and MIP-1α have been shown to be almost identical.Only a difference in the nature of a side-chain or charge interaction inthe vicinity of Trp-57 is observed for the two proteins. Despite havinga similar secondary structure to LD78 and MIP-1α, near u.v. c.d. studiesshow ACT2 has a different tertiary conformation as highlighted by theshape and intensity of the spectrum. This provides strong evidence thatLD78 and not ACT2 is the human homologue of MIP-1α.

A major problem shared by murine MIP-1α and human LD78, which limitstheir potential clinical utility, is that at concentrations as low as 25μg/ml in physiological ionic strength buffer they form large solublemultimeric complexes which have a tendency to aggregate. The nativeMIP-1α and LD78 protein molecules have a molecular weight of 7,866 Daand 7,712 Da respectively. For both proteins, the soluble multimericcomplexes show a broad heterogeneous mixture of molecular weightsranging from 100,000 Da to >>200,000 Da. The principal consequence ofthe multimerisation and aggregation phenomena is that clinicaladministration of the protein is compromised. Aggregation andmultimerisation can lead to varying efficacy, impaired tissuepenetration and enhanced immunogenicity. Another important shortcomingis that, during production and formulation, aggregation will result inheterogeneous pharmaceutical preparations.

Cloudy aggregates are often observed upon reconstitution of pure,lyophilised LD78 or MIP-1α protein in physiological ionic strengthbuffer at pH 7.4. Aggregates are removed by centrifugation prior tofurther analysis. Size exclusion chromatography (Comparative Example 3)of the soluble reconstituted MIP-1α and LD78 following clarificationshow that the majority of the protein chromatographs as broad peaks ofmolecular weights of 100,000-800,000 Da. The broad trailing edge of thepeaks demonstrates the existence of a range of high molecular weightcomplexes for each protein. The size exclusion profile of MIP-1α alsoreveals a population of tetrameric molecules in equilibrium with thelarge multimers.

Although the problem of aggregation of stem cell inhibitors (and LD78 inparticular) has been recognised in the art, those working in the fieldhave hitherto attempted to address it by formulating unusual bufferingsystems (Mantel et al., Expt. Haematol. 20: No. 368 800 (1992), Dunlopet al., Blood 79:2221-2225 (1992) and Graham & Pragnell Dev. Biol. 151377-81 (1992)) to keep the otherwise multimeric molecule in a lowmolecular weight form. This approach has its disadvantages, not leastthat, whatever the ingredients of the buffer, the molecule may wellreaggregate on administration in vivo.

The present invention approaches the problem in a radically differentway. It has been discovered not only how stem cell inhibitors such asLD78 and MIP-1α aggregate but also that it is possible to inhibit theaggregation or multimerisation at certain stages in the aggregationprocess, while still retaining biological activity.

MIP-1α, LD78 and ACT-2 show sequence homology to the chemotacticcytokine superfamily of proteins which contains Interleukin-8 (IL-8),platelet factor 4 (PF-4) and monocyte chemo-attractant and activatingprotein (MCAF). Of these related proteins, it is known that IL-8 existsas a dimer (Clore et al., Biochemistry 29 1689-1696 (1990)) and PF-4 isa tetramer at physiological ionic strength (Moore et al, Biochim.Biophys. Acta. 379, 379-384, 1975). The model of MCAF built using IL-8as a template (Gronenborn & Clore, Protein Engineering 4 263-269 (1991))is consistent with a basic dimeric structure. However, trimerisation oftetramers to dodecamers has not previously been reported.

It has now been found that the high-order multimers of MIP-1α and LD78are formed via intermediate dimers, tetramers and dodecamers (threeassociated tetramer units). So to prevent the undesirable higher ordermultimerisation and aggregation, it is necessary to preventmultimerisation either at the level of or lower than the dodecamer. Atits broadest, therefore, in a first aspect the invention provides aproteinaceous molecule with stem cell inhibition (SCI) activity, themolecule being substantially incapable at physiological ionic strengthof forming a stable multimer higher than a dodecamer. The molecularweight of molecules in accordance with the invention will generally beabout or less than 100,000 Da at physiological ionic strength. Suchvariants would require no further formulation, at least in respect ofthe multimerisation properties, and would therefore represent a clinicaladvantage in terms of ease of use and a manufacturing advantage as itshomogeneity would better lend itself to GMP. Additionally, the increasedmolecular surface area may lead to advantages in tissue penetrabilityand increased efficacy.

Preferred embodiments of the invention are substantially incapable atphysiological ionic strength of forming a stable multimer higher than atetramer; the molecular weight in such cases will generally be about orless than 32,000 Da. Some embodiments are substantially incapable atphysiological ionic strength of forming a stable multimer higher than adimer; in these cases, the molecular weight will generally be about orless than 16,000 Da. Certain embodiments of the invention aresubstantially incapable of forming multimers at all; their molecularweight will generally be about or less than 8,000 Da, which is themonomeric molecular weight of LD78 and MIP-1α, based on amino acidsequences.

Molecules which form substantially homogenous populations of multimers(or monomers) are preferred.

The molecular weight and/or degree of multimerisation of molecules ofthe invention can be assessed by any suitable means. Electrophoresis(for example native PAGE), size-exclusion chromatography and,particularly, ultracentrifuge sedimentation coefficient analysis aremethods of choice.

When it is stated in this specification that a molecule is"substantially incapable" of forming a multimer higher than a givenorder, it should be understood that a minor proportion of higher ordermultimers can be tolerated and may in fact be inevitable from aconsideration of thermodynamic equilibria. While it is not possible toput precise quantitative limits on this proportion, in general no morethan 15, 10 or even 5% of the species present will be above thethreshold stated.

The term "stem cell inhibition activity" (or "SCI activity") is known tothose skilled in the art. It may be taken to refer to a biologicalactivity exhibited by MIP-1α and/or LD78 and in particular to theinhibition of proliferation of stem cells or, more precisely, to theprevention of movement of stem cells through a proliferative cell cycle.Proteinaceous molecules in accordance with the invention may thereforebe regarded as analogues of MIP-1α and/or LD78. "Stem cells", asgenerally indicated above, are dividing cells which maintain cells ofvarious lineages, particularly cells of the haemapoietic system or theepithelial system; more particularly, haemapoietic stem cells are cellswhich are capable of self renewal and are capable of giving long termrepopulation of cells of the haemapoietic system when transplanted intoa lethally irradiated animal.

Stem cell inhibition activity can be determined experimentally in avariety of ways. For example, an in vitro assay of activity can be made.Such an assay is preferably a receptor binding assay: molecules inaccordance with the invention are assessed for their ability to displaceLD78 (or MIP-1α), which may be appropriately detectably labelled, from asuitable source of receptors, such as the murine stem cell line FDCPcell mix (A4 cells). Details of such an assay are given in Example 164below; stem cell inhibition activity may be said to be exhibited by amolecule if a statistically significant proportion of wild type activity(for example at least 1%, 5%, 10%, or 20%, in increasing order ofpreference, of the activity of the corresponding wild type molecule) isobserved for a preparation of given concentration. Receptor bindingactivity which is as good as, or even better than, wild type is notessential but may well be preferred.

An alternative but functional, although still in vitro assay forexperimental determination is an assay which measures the inhibition ofproliferation of murine day 12 CFU-S cells. Molecules are assayed fortheir ability to inhibit colony formation of day 12 CFU-S cells sortedfrom murine bone marrow. Details can be found in:

Lord and Spooner Lymphokine Research B 59 (1986) and Lord and Marsh in"Haemapoiesis, A Practical Approach" IRL Press, Oxford, 1992, Testa andMolineux, Eds., page 21 (for murine bone marrow cell sorting);

Heyworth and Spooncer in "Haemapoiesis, A Practical Approach" IRL Press,Oxford, 1992, Testa and Molineux, Eds., page 37 (for general cellculture techniques); and

Pragnell et al. Blood 72 196 (1988) (for assay and conditioned medium).

More precise details of such an assay are given in Example 165 below.Molecules possess stem cell inhibition activity if they inhibit colonyformation in this assay. Inhibition which is as good as or even betterthan wild type is not essential but may be preferred.

A further alternative functional, but still in vitro, assay is to befound in WO-A-9104274, Pragnell et al., Blood 72 196-201 (1988) andLorimer et al. Leukemia Research 14 481-489 (1990).

Alternatively or additionally, activity in vivo can be assessed. First,a CFU-S in vivo assay, in which the ability of the candidate stem cellinhibitor is used to protect the stem cell population (measured asCFU-S) against the cytotoxic effects of chemotherapeutic agents such ashydroxyurea or cytosine arabinoside (ara-C). A suitable assay isdescribed in Lord et al., Blood 79:2605-2609 (1992) and also by Lord in"Haemopoiesis--A Practical Approach", pages 1-20, IRL Press, Oxford,1992, (Testa and Molineux, Eds.), pages 1-20 and by Lord and Schofieldin "Cell Clones: Manual of Mammalian Cell Techniques", ChurchillLivingstone, 1985 (Potten and Henry, Eds.), pages 13-26.

Further in the alternative, or in addition, an assay which indirectlyreflects the CFU-S population measures the recovery of neutrophilnumbers following chemotherapy with ara-C; such an assay is described inDunlop et al., Blood 79:2221-2225.

A molecule can be regarded as having stem cell inhibiting activity if itgives a significant improvement over negative control (whether or not animprovement over the wild type molecule) in any or all of the aboveassays. Certain preferred molecules show such an improvement overnegative control in more than one, or even all, of the assays.

The term "physiological ionic strength" is well known to those skilledin the art. It is generally equivalent to about 137 mM NaCl, 3 mM KCland about 10 mM phosphate. Physiological pH is about 7.4.

The invention enables the preparation of analogues of LD78 and MIP-1α indodecameric, tetrameric, homo-dimeric and monomeric forms, wherein eachform is substantially incapable of forming a stable higher order complexunder conditions of physiological ionic strength and pH. The analoguesmay form a substantially homogeneous population of multimers; analogueswhich form a substantially homogeneous preparation of tetramers arepreferred.

The term "analogue" is used, broadly, in a functional sense. As apractical matter, though, most analogues will have a high degree ofhomology with the prototype molecule if biological activity is to besubstantially preserved. It will be realised that the nature of changesfrom the prototype molecule is more important than the number of them.As guidance, though, at the amino acid level, it may be that (inincreasing order of preference) at least 40, 50, 60, 65, 67 or 68 of theresidues will be the same as the prototype molecule; at the nucleic acidlevel, nucleic acid coding for an analogue may for example hybridiseunder stringent conditions (such as at approximately 35° C. to 65° C. ina salt solution of approximately 0.9 molar) to nucleic acid coding forthe prototype molecule, or would do so but for the degeneracy of thegenetic code.

Many analogue MIP-1α and LD78 molecules of this invention reproduciblyform a stable quaternary structure no greater than either a tetramer orthree associated tetramer units (a dodecamer). It may well be that thestability of dimers, tetramers, dodecamers or other multimers will varydepending on the environment of the molecules; if so, it will bepreferably at physiological ionic strength and more preferably when theanalogue is presented in a clinically administrable (usually aqueous)formulation, and at a clinically acceptable dose, that multimerisationbeyond a dodecamer cannot (or at least does not) substantially occur.Often the clinically administrable formulation will be reconstitutedfrom a lyophilised protein preparation. Preferably, multimers beyonddodecamers will not substantially occur in conditions likely to beencountered during production, formulation and administration. Theabsence of multimers of molecular weight greater than a tetramer ordodecamer reduces the aggregation of recombinant or other analogues ofMIP-1α and LD78. Such stable preparations of MIP-1α and LD78 analogueswith a defined, reproducible quaternary structure represent a distinctadvantage in production, formulation and administration of thetherapeutic entity.

A stable monomeric, dimeric, tetrameric or dodecameric variant may haveimproved pharmaceutical and pharmacokinetic properties, such as: theadvantage of improved tissue penetration; a lesser likelihood of beingimmunogenic; and much more reproducible efficacy, by virtue of a stablequaternary structure. An added advantage of this approach lies in thefact that some of these surface residues may be involved in receptoractivation and modify the pharmacology. Identification of biologicallyimportant residues can therefore be used to improve the pharmacokineticsof stem cell proliferation inhibition and lead to the design of lowmolecular weight mimics. Stable monomers, dimers, tetramers anddodecamers should provide powerful research tools, being particularlyuseful in the identification and characterisation of receptors for SCIs,of which little is known (Oh et al., J. Immunol. 147 2978-2983 (1991)).Disruption of the dimer interface interactions to produce a monomerwould provide a useful research tool. A further advantage of thisapproach is the possibility of eliminating any cross reaction of LD78 orMIP-1α with the murine inflammatory protein-1β receptor or its humanequivalent. Activation of the MIP-1β receptor elicits a major part ofthe inflammatory response of the body to these molecules and representsa potential unwanted side-effect during therapy. Elimination of thisresponse would therefore provide a further clinical advantage.

An unexpected additional advantage conferred by the lack ofmultimerisation is the greatly enhanced productivity of such variants ineukaryotic cells, for example yeast species such as Saccharomycescerevisiae and Pichia pastoris. The invention therefore relatesadditionally to a method of increasing protein expression levels in asystem in which the desired protein normally forms multimeric complexes(which may be soluble) at physiological ionic strength (the "multimericprotein"), which method comprises using in the expression system cellswhich are transformed or transfected with a vector comprising DNAcoding, not for the multimeric protein, but instead for a mutant thereofwhich has a reduced tendency to form (eg soluble) multimeric complexesrelative to the multimeric protein. Such a method may be of generalapplicability, but has particular utility when applied to the productionof proteins having stem cell inhibitor activity.

From studies involved in the making of this invention, it appears thatLD78 and MIP-1α associate along the following pathway:

    M+M←→D; D+D←→T; 3×T←→dodecamer;

    n×dodecamer←→multimer; n×multimer→aggregate.

wherein M represents a monomer, D represents a homo-dimer and Trepresents a tetramer. FIGS. 1c and 1d illustrate how this putativelycomes about. Circumstantial evidence in support of this proposal comesfrom Mayo and Chen (Biochemistry 28 9469-9478 (1989)), who demonstratedthat tetramers of PF-4 form via a similar pathway.

The pathway proposed above consists of a series of reversible equilibriaup to the point of the irreversible aggregation of multimers. There arein principle four stages in the association mechanism at which it ispossible to prevent the formation of large multimers (and thereforeaggregates) of SCIs. Inhibition of each of these stages could beinfluenced by a mutation in a different region of the SCI molecule.

First, further association of tetramers can be inhibited. Secondly, ifthe SCI dimers are prevented from associating to tetramers, then furthermultimerisation will be inhibited. Thirdly, SCI monomers may beprevented from dimerising. Fourthly, further association of dodecamersto higher order multimers can be inhibited. Any of these options can beimplemented by specific mutation of residues involved in promotingand/or stabilising the association events. A further option would be touse a combination of mutations simultaneously to block two or all of theassociation events.

The following amino acid residues are preferred for modification:

(i) amino acid residues which could be involved in stabilising theinteraction between two dimers; and

(ii) amino acid residues at surface regions, on the external faces ofthe tetramer, which could act as sites for higher order association.

Radical mutation of individual or combinations of key residuesstabilising the association of dimers into tetramers will yield adimeric recombinant SCI variant or analogue molecule. Similarly,mutation of residues at the sites of association of tetramers tomultimers will yield a tetrameric SCI variant or analogue molecule. Theamino acid modification preferably involves a substitution, althoughdeletions and additions are contemplated within the scope of theinvention.

The types of mutation preferred for producing the desired effects are:

(i) charge repulsions (successfully used to produce monomeric insulin;Dodson, Prospects in Protein Engineering Meeting Abstracts, 49-53,(1989));

(ii) hydrophobic to hydrophilic changes;

(iii) neutral/hydrophobic to charged.

It is generally better not to substitute very hydrophobic residues intothe protein in order to avoid contributing to the hydrophobic effect inassociation. Equally, it is preferred to avoid mutations whichsignificantly disrupt secondary structural elements of the protein: so,for example, known β-breakers are preferably not introduced into β-sheetregions.

Certain types of mutation are most effective in producing desirablechanges within the SCI molecule. These are:

charge reversal;

charged residue to neutral;

hydrophobic to hydrophilic.

For optimum results substitutions should be made at particular siteswithin the molecule. The residues which should be altered are dependenton the level of multimerisation which is to be prevented.

The following discussion of preferred sites for mutation deals primarilywith LD78, the proposed structure of which is shown in FIG. 1b. In FIG.1b, the ribbon traces the predicted path of backbone atoms for the LD78monomer. The labelled residues define the putative secondary structureelements. β-sheet strand 1 runs from Phe23 to Thr30; β-sheet strand 2runs from Lys35 to Thr43; β-sheet strand 3 runs from Ser46 to Pro53; andthe C-terminal helix runs from Trp57 to Ala69. Analogous secondarystructural elements may be inferred for other SCIs, including MIP-1α,for example using the amino acid alignment shown in FIG. 1a.

It is apparent that some faces of the monomer are involved in more thanone part of the multimerisation pathway. The extent ofdisruption/inhibition of self-association in those faces is related tothe nature of the amino acid substitution.

Inhibition of monomer to dimer formation can be achieved by one or moremutations, for example at residue 19 (Ile) or 39 (Val). Either residuemay be changed to Ala.

Dimer to tetramer formation is affected by mutations in residuesprojecting away from the surface of the dimer in strand 1 of the βsheet, and/or in the turn between strands 2 and 3 of the sheet. Examplesof the first region are amino acids 24-29 of LD78 and of the secondregion are amino acids 43-47 of LD78. In particular, Ile24>Asn,Tyr27>Asn, Phe28>Glu, Glu29>Arg, Lys44>Glu (especially with Arg45>Gln)and Arg 45>Glu are preferred.

Tetramer to dodecamer formation can be inhibited or disrupted bymutations of the nature described above in either the residues whichform a chain N-terminal to the turn into strand 1 of the sheet (wheretwo changes are preferred), particularly residues 16-21, especially17-19 or at position 4, 12, 26, 44, 48 or 66 of LD78. In particular,Ala4>Glu, Phe12>Asp, Arg17>Ser, Asp26>Ala (especially with Gln18>Glu),Arg17>Glu (again especially with Gln18>Glu), Asp26>Ala, Lys44>Ser,Gln48>Glu (especially with Phe28>Glu) and Glu66>Ser are preferred.

Dodecamer to higher order multimer formation is prevented or disruptedby mutations at positions 12 to 21, especially positions 12, 18 and 21,of LD78, or at position 65. In particular, Phe12>Gln, Gln18>Glu,Gln21>Ser and Leu65>Ala are preferred.

Generally preferred LD78 analogues of the invention include moleculeswhich comprise a sequence substantially corresponding to LD78, but witha mutation at one or more (but preferably no more than two) of thefollowing amino acid residues: Ser1, Leu2, Ala3, Ala4, Asp5, Thr6, Ala9,Phe12, Ser13, Tyr14, Ser16, Arg17, Gln18, Ile19, Pro20, Gln21, Phe23,Ile24, Asp26, Tyr27, Phe28, Glu29, Ser31, Ser32, Gln33, Ser35, Lys36,Pro37, Gly38, Val39, Ile40, Leu42, Thr43, Lys44, Arg45, Ser46, Arg47,Gln48, Asp52, Glu55, Glu56, Gln59, Lys60, Tyr61, Val62, Asp64, Leu65,Leu67, Glu66, Ser68, and Ala69.

Preferred LD78 analogues in accordance with the invention includeLys44>Glu (with Arg45>Gln), Arg47>Glu, Phe28>Glu, Phe28>Glu (withGln48>Glu), Phe28>Glu (with Arg47>Glu), Arg17>Ser (with Gln18>Glu),Phe12>Ala, Val39>Ala, Ile40>Ala, Asp26>Ala (with Glu29>Arg andArg47>Glu). More preferred LD78 analogues in accordance with theinvention include Arg17>Ser, Glu29>Arg, Gln18>Glu, Asp26>Ser, Gln48>Ser,Thr15>Ala, Gln21>Ser, Phe23>Ala, Ser32>Ala, Ala51>Ser, Ala4>Glu,Phe12>Asp, Asp26>Gln, Lys36>Glu, Lys44>Glu, Arg45>Glu, Glu66>Gln. Themost preferred LD78 analogues in accordance with the invention arePhe12>Gln, Lys44>Ser, Arg17>Glu (with Gln18>Glu) and, especially,Asp26>Ala and Glu66>Ser.

Generally preferred MIP-1α analogues of the invention include moleculeswhich comprise a sequence substantially corresponding to MIP-l1α, butwith a mutation at one or more (but preferably not more than two) of thefollowing amino acid residues: Ala1, Pro2, Tyr3, Gly4, Ala5, Asp6, Thr7,Ala10, Phe13, Ser14, Tyr15, Ser16, Arg17, Lys18, Ile19, Pro20, Arg21,Phe23, Ile24, Asp26, Phe28, Glu29, Ser31, Ser32, Leu33, Ser35, Gln36,Pro37, Gly38, Val39, Ile40, Leu42, Thr43, Lys44, Arg45, Asn46, Arg47,Gln48, Asp52, Glu55, Thr56, Gln59, Glu60, Tyr61, Ile62, Asp64, Leu65,Glu66, Leu67, Asn68 and Ala69.

Preferred MIP-1α analogues of the invention correspond to the preferredLD78 analogues described above.

Molecules in accordance with the invention will for preference be freeof N-terminal extensions preceding Ser-1 (in the case of LD78) or Ala-1(in the case of MIP-1α). This is because such N-terminally extendedforms of the molecule are compromised with respect to their ability tobind to the LD78 receptor present on stem cells. Such molecules canstill give rise to active species in functional in vitro assays, such asCFU-A or mitogenesis assays, possibly due to processing byaminopeptidases. It is preferable, however, not to depend on suchprocessing events for the clinical application of a stem cell inhibitor,as it leads to greater uncertainty over the pharmacokinetics of theactive species, and increased variation in the response.

In contrast to the N-terminally extended variants, molecules carryingN-terminal deletions of between 1 and 7 residues are active in receptorbinding, though the full length form with serine at position 1 ispreferred.

SCI analogues in accordance with the invention can in principle be madeby any convenient method including chemical modification of existing(for example natural) proteins and/or chemical coupling of two or moreoligo- or polypeptide chains. Far greater flexibility, though, can beobtained by using recombinant DNA methodology, which enables successiveamino acid residues to be coupled together in vivo.

According to a second aspect of the invention, therefore, there isprovided nucleic acid coding for a protein as described above. Both DNAand RNA are within the scope of the invention. DNA may be chemicallysynthesised and/or recombinant.

Mutations may be introduced by de novo polynucleotide synthesis, bysite-directed mutagenesis using appropriately designed oligonucleotideprimers or by any other convenient method.

Recombinant DNA in accordance with the invention may be in the form of avector. The vector may for example be a plasmid, cosmid or phage.Vectors will frequently include one or more selectable markers to enablethe selection of cells transformed (or transfected: the terms are usedinterchangeably in this specification) with them and, preferably, toenable selection of cells harbouring vectors incorporating heterologousDNA. Appropriate translational initiating and termination signals willgenerally be present. Additionally, if the vector is intended forexpression, sufficient transcriptional regulatory sequences to driveexpression will be included. Vectors not including regulatory sequencesare useful as cloning vectors.

Cloning vectors can be introduced into E. coli or any other suitablehosts which facilitate their manipulation. Expression vectors may beadapted for prokaryotic expression but for preference are adapted forexpression in a microbial eukaryotic cell, such as a yeast (includingbut not limited to Saccharomyces cerevisiae and Pichia pastoris) or ahigher eukaryotic cell such as insect or mammalian cells.

Performance of the invention is neither dependent on nor limited to anyparticular strain of microorganism or cell type: those suitable for usewith the invention will be apparent to those skilled in the art,following the teaching of this specification. According to a thirdaspect of the invention there is provided a host cell transfected ortransformed with DNA described above. Host cells may be of any suitablesource; eukaryotic host cells are preferred; yeast cells may be those ofchoice.

Production of stem cell inhibitors, whether wild type or altered asdescribed above has been found to be particularly advantageous whencarried out in the yeast host Pichia pastoris. According to a fourthaspect of the invention, therefore, there is provided a process for theproduction of a molecule having stem cell inhibitor activity, theprocess comprising culturing a yeast of the genus Pichia, and preferablyof the species pastoris, the yeast having expressible heterologousnucleic acid coding for the molecule.

DNA in accordance with the invention can be prepared by any convenientmethod involving coupling together successive nucleotides, and/orligating oligo- and/or poly-nucleotides, including in vitro processes,but recombinant DNA technology forms the method of choice.

However the proteinaceous compounds of the invention are made, they maybe useful either as research tools or in medicine. Other uses are notruled out. In a fifth aspect, the invention provides proteinaceouscompounds as described above for use in medicine, particularly in theprotection of stem cells, for example in tumour therapy (whetherradiotherapy or chemotherapy).

The invention therefore provides in a sixth aspect the use of aproteinaceous compound as described above in the preparation of an agentfor use as a stem cell protective agent, particularly in tumour therapy.The invention can be used in a method for the protection of stem cells,particularly in conjunction with tumour therapy, the method comprisingadministering to a patient an effective amount of a proteinaceouscompound as described above. This method is preferably performed invivo. Alternatively, the method may be performed ex vivo where theresulting marrow is purged of leukaemia cells by the chemotherapeuticagent and then reinjected into the patient.

Formulations of the proteinaceous compounds described above themselvesform an aspect of the invention and comprise active compound and apharmaceutically acceptable carrier. While oral formulations which leadto bioactive and bioavailable active compound may in principle bepreferred, in practice the compounds of the invention may have to beadministered parenterally. Parenterally administrable formulations willgenerally be sterile and may comprise one or more proteinaceouscompounds dissolved in a suitable liquid excipient such as water forinjections, PBS or physiological saline. Dosages will be determinable bythe clinician or physician and will generally be such as to ensure anactive dose is delivered.

Compounds of the invention may also be used to treat psoriasis or otherdisorders related to hyper-proliferative stem cells either alone or inconjunction with cytotoxic agents. Topical or transdermal formulationsof compounds of the invention, as well as parenteral formulations, mayadvantageously be used in this aspect of the invention.

It is to be understood that preferred features for each aspect of theinvention are as for each other aspect of the invention, mutatismutandis.

Certain preferred embodiments of the invention will now be describedwith reference to the accompanying drawings, in which:

FIG. 1a illustrates the alignment of the LD78 (SEQ ID NO. 2) amino acidsequence with those of MIP-1α (SEQ ID NO. 18) and ACT-2; (SEQ ID NO. 35)

FIG. 1b shows the structural model of the LD78 monomer. The ribbontraces the predicted path of the backbone atoms for the LD78 monomer.The labelled residues define the predicted secondary structure elements.Strand 1 of the β-sheet is from Phe23 to Thr30, strand 2 is from Lys35to Thr43, strand 3 is from Ser46 to Pro53 and the C-terminal helix isfrom Trp57 to Ala69.

FIG. 1c shows schematically the putative multimer interfaces on the LD78monomer.

FIG. 1d shows how the LD78 monomer shown in FIG. 1c are proposed to formdimers, tetramers, dodecamers and aggregates.

FIG. 2 illustrates the plasmid of yeast expression vector pSW6.

FIG. 3 demonstrates that the tertiary conformation of LD78 and MIP-1αare identical as determined by near ultra-violet circular dichroism.

FIG. 4 demonstrates that the tertiary conformation of ACT2 differs fromthat of MIP-1α as determined by near ultra-violet circular dichroism.

FIG. 5 illustrates a western blot of MIP-1α, LD78 and ACT2 withanti-MIP-1α antibody. The result demonstrates cross-reaction of LD78 butnot ACT2 with anti-MIP-1α.

FIG. 6 shows a representative size exclusion chromatographic profile ofMIP-1α, LD78 and ACT2, reconstituted as described in Comparative Example4. Also shown is the elution profile of proteins used as standard ofmolecular weights demonstrating the correct separation of standardproteins and a table of molecular weight species determined forreconstituted LD78, MIP-1α and ACT-2.

FIG. 7 shows a Coomassie-stained native PAGE analysis of LD78, MIP-1αand ACT-2 with mixed molecular weight markers and EGF standard. The geldemonstrates that the three proteins all run as high molecular weightmultimers under native conditions.

FIG. 8 shows the distribution of protein solute mass in the analyticalultracentrifuge cell at equilibrium for LD78 wild type in various bufferconditions.

FIG. 9 shows the near u.v. circular dichroism spectra of tetrameric LD78is independent of the buffer conditions used to achieve the definedstructure.

FIG. 10 shows that the near u.v. c.d. spectrum of tetrameric LD78 isdifferent to that of the high molecular weight multimer form.

FIG. 11 shows that quenching of Trp-57 fluorescence emission energyoccurs is present in the multimeric complexes but not in the tetramer.This quenching of emission energy arises due to the presence of anelectrostatic interaction unique to the multimers which is proximal toTrp-57.

FIG. 12 shows a coomassie stained native PAGE gel with wild type LD78,mutant 10, 11, 52 and mixed molecular weight markers. The geldemonstrates the different electrophoretic mobilitie observed for LD78variants. Mutant 10 is the subject of Example 7, mutant 11 is thesubject of Example 8 and mutant 52 is the subject of Example 64.!

FIG. 13 shows a Coomassie blue stained native PAGE gel with native LD78,7 mutant constructs and mixed molecular weight markers. The geldemonstrates that mutants 1, 2, 10, 15, 26, 29 and 30 (of Examples 1, 2,7, 11, 16, 19 and 20, respectively) have different multimerisationproperties from wild type exhibiting (to different extents) fasterelectrophoretic mobility.

FIG. 14 shows the size exclusion chromatography (SEC) profiles in 150 mMPBS pH7.4 of selected mutant constructs with wild type LD78 forcomparison.

FIG. 15a shows the Pichia pastoris expression vector pHILD4.

FIG. 15b shows the Pichia pastoris expression vector pLHD4 whichincludes an EGF gene fused to the a factor preprosequence.

FIG. 16 shows the Pichia pastoris expression vector pHILD 1.

FIG. 17 shows the Pichia pastoris expression vector pLH23, which isbased on pLHD4 modified to direct expression and secretion of LD78.

FIG. 18 shows the construction of the optimised Pichia pastoris LD78secretion vector pLH23.

FIG. 19 shows the correlation between multimerisation state and receptorbinding.

FIG. 20 shows a computer-generated model of the proposed LD78 tetramerwith receptor binding residues highlighted in black.

FIG. 21 shows the annealed oligonucleotides for the construction of LD78synthetic gene (SEQ ID NOS 4-13) with overlapping cohesive ends.

FIG. 22 shows the annealed oligonucleotides used in the construction ofthe MIP-1α gene (SEQ ID NOS 20-31) with overlapping cohesive ends.

FIG. 23 shows the annealed oligo nucleotides used in the construction ofthe ACT-2 gene (SEQ ID NOS 37-46) with overlapping cohesive ends.

FIG. 24 shows the effect of mutant 10 on in vitro colony formation usingpurified murine stem cells.

FIG. 25 shows the effect of mutant 82 on in vitro colony formation usingpurified murine stem cells.

Preparations 1-14 describe the construction of synthetic genes for LD78,MIP-1α and ACT-2, the development of yeast expression vectors and theproduction and preliminary characterisation of their protein products.

Comparative Examples 1-7 describe the biophysical properties of LD78,MIP-1α and ACT-2, a comparison of their molecular weight under a rangeof solution conditions and a spectroscopic assay for the extent of LD78multimerisation.

Examples 1-124 describe the design and construction of LD78 variants andtheir incorporation into expression vectors. Example 125 discloses aconvenient gel screen for the detection of variants with alteredmulti-merisation properties. Examples 126-153 describe the effect onmultimerisation of mutations at particular residues. Example 154 showsthat previously described variants of LD78 are wild-type with respect totheir multimerisation characteristics. Example 155 discloses themolecular faces involved in LD78 multimerisation.

Examples 156-157 disclose the unexpected observation that LD78 variantsexhibiting reduced multimerisation give higher expression levels in S.cerevisiae than wild-type LD78. Examples 158-163 describe theconstruction of an improved Pichia pastoris expression vector, theconstruction of LD78 producing strains and the unexpectedly high yieldsof wild-type LD78 that are obtained, and demonstrate that furtherincreases in yield are observed with variants exhibiting reducedmultimerisation.

Example 164 demonstrates that variants exhibiting reducedmultimerisation are active in an in vitro model of receptor binding.Example 165 shows that demultimerised mutants can inhibit theproliferation of haematopoietic progenitor cells (day 12 CFU-S).

METHODOLOGY

The techniques of genetic engineering and genetic manipulation used inthe manufacture of the genes described and in their further manipulationfor construction of expression vectors are well known to those skilledin the art. Descriptions of modern techniques can be found in thelaboratory manuals "Current Protocols in Molecular Biology", Volumes 7and 2, edited by F. M. Ausubel et al, published by Wiley-Interscience,New York and in "Molecular Cloning, A Laboratory Manual" (secondedition) edited by Sambrook, Fritsch and Maniatis published by ColdSpring Harbor Laboratories, New York. M13mp18, M13mp19 and pUC18 andpUC19 DNAs were purchased from Pharmacia Ltd., Midsummer Boulevard,Central Milton Keynes, Bucks, MK9 3HP, United Kingdom. Restrictionendonucleases were purchased either from Northumbria BiologicalsLimited, South Nelson Industrial Estate, Cramlington, Northumberland,NE23 9HL, United Kingdom or from New England Biolabs, 32 Tozer Road,Beverly, Mass. 01915-5510 USA.

Preparation 1--Construction of a Synthetic Gene for Human LD78

Gene Design

The published amino acid sequence for LD78 was reverse-translated togive a gene sequence. The codon usage was then optimised to maximiseexpression in S. cerevisiae. The 5' end of the synthetic gene wasdesigned to include codons for the last five amino acid residues (SerLeu Asp Lys Arg) of the yeast mating type factor alpha. The sequence wasthen modified to include a HindIII restriction site at the 5' end and aBamHI restriction site at the 3' end (SEQ ID: 1).

The gene sequence was divided into 12 oligonucleotides (SEQ ID:4 to SEQID: 13). Each internal oligonucleotide was designed so that a unique 7base cohesive end is left after anealling each pair of complementaryoligonucleotides. This allows for perfect oligo matching during geneconstruction. FIG. 21 shows the annealed oligonucleotides withoverlapping cohesive ends.

Oligonucleotide Synthesis

The oligonucleotides were synthesised on an Applied Biosystems 380B GeneSynthesiser, using cyanoethyl phosphoramidite chemistry. The methodologyis now widely used and has been described (Beaucage, S. L. andCaruthers, M. H. Tetrahedron Letters 24. 245 (1981)).

Gene Construction

In order to create a full length gene, 100 pmole of each oligonucleotidewas dried down in a vacuum dessicator. The 5' ends of internaloligonucleotides were kinased to provide a 5' phosphate to allowsubsequent ligation. 100 pmoles of dried oligomer was resuspended in 20μl of kinase buffer (70 mM Tris, pH7.6, 10 mM MgCl₂, 1 mM ATP, 0.2 mMspermidine, 0.5 mM dithiothreitol). T4 polynucleotide kinase (2 μl.10,000 U/ml) was added and the mixture was incubated at 37° C. for 30minutes. The kinase was then inactivated by heating at 70° C. for 10minutes. Note that end oligonucleotides BB5615 and BB5624 were notkinased to prevent concatemerisation during the construction (SEQ ID:2).

Complementary pairs of oligonucleotides were annealed in single pairs(90° C., 5 minutes, followed by slow cooling to room temperature). The 6annealed pairs were then mixed together, heated at 50° C. for 5 minutes,and ligated overnight at 14° C. using T4 DNA ligase. The ligated fulllength product was then separated from non-ligated material byelectrophoresis on a 2% low melting temperature agarose gel. The DNAfragment corresponding to the LD78 gene was excised and extracted fromthe gel. The purified fragment was then ligated to HindIII and BamHItreated pUC18 plasmid DNA. The ligated products were transformed into asuitable E. coli host strain using standard methodology. The strain usedwas HW87 which has the following genotype:

araD139Δ(ara-leu) 7697Δ(lacIPOZY)74 galU galK hsdR rpsL srl recA56

The use of this particular strain is not critical: any suitablerecipient could be used (eg MC1061, available from the American TypeCulture Collection (ATCC)). Transformants were selected on L-agarcarbenicillin plates. Twelve carbenicillin-resistant colonies werepicked and used to prepare plasmid DNA for sequence analysis. Doublestranded dideoxy sequence analysis using a universal sequencing primer(United States Biochemical Corporation, (5'-GTTTTCCCAGTCACGAC-3' (SEQ IDNO 14)), was used to identify a correct clone pUC18-LD78. The pUC18-LD78vector was used as a source of the LD78 gene to construct the expressionvector.

Preparation 2--Construction of a Yeast Expression Vector for Human LD78

An expression vector was designed to enable secretion of LD78 to theextracellular medium after expression in S. cerevisiae. Secretion aidspurification and rapid analysis of LD78. The secretion signals from theyeast mating type factor alpha were used to direct export of the LD78protein.

The yeast expression vector pSW6 (SEQ ID NO 15, FIG. 2) is based on the2 micron circle from S. cerevisiae. (pSW6 was deposited in S. cerevisiaestrain BJ2168 at the National Collection of Industrial and MarineBacteria Limited, 23 St. Machar Drive, Aberdeen AB2 1RY, Scotland UnitedKingdom on 23rd Oct. 1990 under Accession No. NCIMB 40326.) pSW6 is ashuttle vector capable of replication in both E. coli and S. cerevisiaeand contains an origin of DNA replication for both organisms, the leu 2gene (a selectable marker for plasmid maintenance in the yeast host) andthe ampicillin resistance locus for selection of plasmid maintenance inE. coli. (The DNA sequence for the vector has been determined; the E.coli sequences are derived from the E. coli ColE1-based repliconpAT153.) The full sequence is given in SEQ ID NO 15. The ability topassage this vector through E. coli greatly facilitates its geneticmanipulation and ease of purification. pSW6 contains an alpha-factorpre-pro-peptide gene fused in-frame to a gene encoding human epidermalgrowth factor (EGF). The expression of this fusion is under the controlof an efficient galactose regulated promoter which contains hybrid DNAsequences from the S. cerevisiae GAL 1-10 promoter and the S. cerevisiaephosophoglycerate kinase (PGK) promoter. Transcription of the EGF geneis terminated in this vector by the natural yeast PGK terminator. TheEGF gene in pSW6 can be removed by digestion with restrictionendonucleases HindIII and BamHI. This removes DNA encoding both EGF and5 amino acids from the C-terminus of the alpha-factor pro-peptide. Genesto be inserted into the pSW6 expression vector must therefore have thegeneral composition: HindIII site-alpha factor adaptor-gene-BamHI site.

After digestion with HindIII and BamfHi endonucleases, the pSW6 vectorcontains the alpha factor gene minus the codons for the last five aminoacid residues. To construct the alpha factor-LD78 fusion gene in thepSW6 vector, the pUC18-LD78 vector of Preparation 1 was treated withHindIII and BamHI endonucleases. The products of this digestion reactionwere separated by electrophoresis on a 1% low gelling temperatureagarose gel. The DNA fragment (ca. 235 bp) corresponding to the LD78gene was excised and purified from the gel matrix. This DNA fragment wasthen ligated into HindIII and BamHI treated pSW6 DNA (the vector DNAfragment lacking the EGF insert was purified for use in this ligation).The recombinant ligation products were transformed into competent HW87E. coli cells. Transformants were selected on L-agar ampicillin plates.12 Ampicillin resistant transformants were screened by preparation ofplasmid DNA and restriction endonuclease analysis with HindIII and BamHIfollowed by agarose gel electrophoresis. A clone (pSW6-LD78) with thecorrect electrophoretic pattern was selected. A plasmid preparation ofthis vector was prepared and the integrity of the construct wasconfirmed by dideoxy sequence analysis on the plasmid DNA usingsequencing primer BB1330 (5'-AGGATGGGGAAAGAGAA-3') (SEQ ID NO: 16). Thisplasmid is the expression vector used for wild-type LD78 expression.

Preparation 3--Expression of Human LD78 Synthetic gene in S. cerevisiae

pSW6-LD78 plasmid DNA of Preparation 2 was prepared and electroporatedinto yeast (S. cerevisiae) strain MC2 which has the following genotype:prc1-407 prb1-1122, pep4-3, leu23-112, trp1 ura3-52 mating type α. Theuse of strain MC2 is not critical for use either in this preparation orin the invention in general. Any suitable strain can be used, such asfor example strain BJ2168, which is genetically almost identical to MC2and is deposited (see Preparation 2 above).

Using the method described in the Bio-Rad manual (GENE PULSER™transfection apparatus, Operating Instructions and Applications Guide,Version 10-90, Bio-Rad Laboratories, 3300 Regatta Boulevard, Richmond,Calif. 94804 USA) the plasmid DNA was electroporated. Briefly, yeaststrain MC2 was grown overnight in YPD medium at 30° C. overnight. Cellswere harvested by centrifugation at 3000 r.p.m for 5 mins in a BeckmanGS-6KR centrifuge, washed in sterile water and resuspended in 1Msorbitol, and then added in 40 μl aliquots to various amounts (0.1 μg-1μg) of plasmid DNA. The resulting mixtures were subjected to a pulse of1500 volts for 5 msec, and added to 300 μl of 1M sorbitol. Theelectroporated cells were then plated out onto agar-sorbitol plates andallowed to grow for 4-5 days at 30° C.

All yeast media were as described by Sherman et al., "Methods in YeastGenetics", Cold Spring Harbor Laboratory, (1986)).

Yeast Expression and Purification

After 4-5 days, electroporatants were picked and plated onto fresh agarplates. Single colonies were obtained after a further 1-2 days growth at30° C. Single colonies were then used to inoculate 5 ml of YPD mediumand the cultures were grown overnight at 30° C. This 5 ml overnightculture was then used to inoculate 0.5 liter shake flasks containing 50ml of 0.67% synthetic complete medium, yeast nitrogen base, with aminoacids minus leucine and 1% glucose as a carbon source and grownovernight at 30° C. After 24 hrs growth, cells were harvested bycentrifugation at 3000 rpm for 5 minutes in a SORVALL™ RC3-B centrifugeand used to inoculate 100 ml of the same synthetic complete medium(except that 1% galactose and 0.2% glucose were used as the carbonsource). This induces gene expression from the hybrid PGK promoter.Induction was carried out by growth in the galactose containing mediumat 30° C. for 48-72 hours.

After either 48 or 72 hrs the culture supernatant was collected bycentrifugation in a SORVALL™ RC3-B centrifuge at 3000 rpm for 5 minutesto remove cells. This supernatant was used for further analysis andpurification of LD78 according to the methods described in Preparation4.

Preparation 4--Purification of human LD78 expressed from a syntheticgene in yeast

Supernatant from the shake flasks described in preparation 3 was spun at6500 rpm (SORVALL™ RC5-B centrifuge) for 15 minutes to clarify.Typically 3 liters of yeast supernatant were adjusted to pH8 and 30 mlof Q-SEPAHROSE™ ion exchange resin (Pharmacia) pre-equilibrated in 50 mMTris pH8.0 was added. Protein was batch adsorbed overnight at 4° C. withgentle agitation. The resin was then allowed to settle and thesupernatant removed. The resin was then poured into a column 1.6 cm indiameter and washed with 10 volumes of 50 mM Tris pH8.0, the protein wasthen eluted in 0.5M NaCl, 50 mM Tris pH8.0 (typically 50 ml total volumeof eluent). The eluent was transferred into prewetted SPECTRAPOR™dialysis membrane (3000 Da cutoff) and dialysed against 10× volume 50 mMTris pH8.0 at 4° C. with one change of buffer. The sample was adjustedto 20% acetonitrile (final concentration) and the pH brought to 3.0 withhydrochloric acid. The protein sample was then pumped directly(bypassing the Rheodyne injection loop) onto a 20 ml VYDAC™ C-18 (10μpore-size) semi-preparative reverse phase HPLC column pre-equilibratedat 3ml/min in 20% acetonitrile, 0.1% trifluoracetic acid (TFA) andeluted with a linear gradient from 20% to 50% acetonitrile, 0.1% TFAover 40 minutes. Eluting fractions were detected by u.v. absorbance at280 nm and analysed by SDS-PAGE PHASTGEL™ (as described in Preparation11). The pure LD78 protein was found to elute around 43% acetonitrile,0.1% TFA. Purified LD78 was freeze-dried and stored at -20° C. Thesequence of the protein is given as SEQ ID NO 2.

Preparation 5--Construction of a Synthetic Gene for Murine MIP-1α

Gene Design

The published amino acid sequence for MIP-1α was reverse-translated togive a gene sequence. The codon usage was then optimised to maximiseexpression in S. cerevisiae. SEQ ID NO 17 shows the sequence of thesynthetic gene, the protein sequence is given as SEQ ID NO 18 and theantisense strand of the gene is SEQ ID NO 19. The method of Preparation1 was followed for the construction of the MIP-1α gene except that theoligonucleotides differ from those in preparation 1. FIG. 22 shows theannealed oligonucleotides used in the construction of the MIP-1α gene(SEQ ID NOS 20-31). The synthetic gene was cloned into plasmid pUC18 tocreate pUC18MIP-1α.

Preparation 6--Construction of a Yeast Expression Vector for MurineMIP-1α

With the exception of the changes detailed below, the method ofPreparation 2 was followed for the construction of a yeast expressionvector designed to enable secretion of MIP-1α from S. cerevisiae.

The MIP-1α synthetic gene in pUC18MIP-1α vector of Preparation 5 must beengineered prior to its inclusion into the pSW6 expression vector. Thisis because the synthetic MIP-1α gene lacks sequences at the 5' endsuitable for the construction of an in frame fusion to the alpha factorgene in the pSW6 vector. To rebuild the DNA encoding the amino acids atthe C-terminal end of the alpha-factor pro-peptide and to fuse this tothe synthetic MIP-1α gene, an oligo nucleotide adapter BB985(5'-AGCTTGGATAAAAGA-3' (SEQ ID 32, top strand), BB986 5'-TCTTTTATCCA-3'(SEQ ID 33, bottom strand)) containing a HindIII site and codonsencoding the Ser, Leu, Asp, Lys and Arg from the C-terminal end of thealpha-factor pro-peptide was constructed. The alpha factor adaptor wasligated to the synthetic MIP-1α gene such that the recombinant geneencoded an in-frame alpha-factor pro-peptide fusion to MIP-1α. ThepUC18MIP-1α plasmid of Preparation 5 was first cleaved with BspMI andthe overhanging ends were filled using DNA polymerase I to create ablunt ended linear DNA fragment. The linearised DNA fragment wasseparated from uncut plasmid DNA on a 1% low gelling temperature agarosegel matrix, then further treated with HindIII. The fragment was thenligated to the alpha-factor adaptor described above. Note that the twostrands of the adaptor were annealed prior to ligation. The recombinantligation products were transformed into competent cells of E. coli HW87.Ampicillin-resistant transformants were analysed by preparation ofplasmid DNA, digestion with HindIII and BamHI and agarose gelelectrophoresis. A correct recombinant plasmid was identified. Theintegrity of this vector was confirmed by dideoxy sequencing analysisusing sequencing primers BB3376 and BB3379. (BB3376 and BB3379 are showntogether in SEQ. ID: 5.)

This plasmid was used as a source of DNA for construction of the yeastexpression vector. The method of Preparation 2 was followed. Briefly themodified pUC18 MIP-1α vector now containing the alpha factor adaptor wasdigested with HindIII and BamHI and the MIP-1α DNA fragment waspurified. This fragment was ligated to HindIII and BamHI treated pSW6DNA according to the method in Preparation 2. The MIP-1α expressionvector, the subject of Preparation 7, was called pSW6MIP-1α. This vectorwas used for subsequent expression.

Preparation 7--Expression of Synthetic Murine MIP-1α in Yeast

The method of Preparation 3 was used for the expression of the murineMIP-1α gene with the exception that the expression vector used waspSW6MIP-1α and transformation was used in place of electroporation. Themethod of Sherman F. et al., ("Methods in Yeast Genetics", Cold SpringHarbor Laboratory, (1986)) was used for transformation.

Preparation 8--Purification of murine MIP-1α expressed from a syntheticgene in yeast

Supernatant from the shake flasks described in Preparation 7 wascentrifuged at 5000 rpm in a SORVALL™ RC-5B centrifuge to clarify.Typically 5 liters of clarified supernatant were adjusted to 20%acetonitrile, 0.1% TFA (final concentration) and 30 g of C-18 silicaresin was added as a dry powder. Protein was batch adsorbed onto theresin overnight at 4° C. with gentle agitation. The silica resin wasthen allowed to settle and the supernatant removed. Resin was thenpoured into a 2.5 cm (diameter) column, washed with 10 column volumes of25% acetonitrile, 0.1% TFA and eluted with 50% acetonitrile, 0.1% TFA;30 ml fractions were collected manually. Aliquots of these fractionswere dried and analysed by SDS-PAGE PHASTGEL™ (Pharmacia) as describedin Preparation 13. The protein concentration was estimated from theabsorbance at 280 nm in a 1 cm pathlength cell and a calculatedabsorbance of 1.37 for a 1 mg/ml protein solution under the sameconditions. The eluted fractions were then freeze-dried. For furtherpurification, the dried fractions were reconstituted in 0.1% TFA (finalconcentration) and 4mg aliquots loaded onto a 20 ml DYNAMAX™semi-preparative C-18 reverse phase column (10μ pore-size) at 3 ml/minequilibrated in 25% acetonitrile, 0.1% TFA. MIP-1α was eluted using alinear 25-45% acetonitrile, 0.1% TFA gradient over 50 minutes. Elutingfractions were detected by u.v. absorbance at 280 nm and collectedmanually. Purified MIP-1α was freeze dried and stored at -20° C.

Preparation 9--Construction of a Synthetic Gene for Human ACT-2

Gene Design

The published amino acid sequence for human ACT-2 was reverse-translatedto give a gene sequence. The codon usage was then optimised to maximiseexpression in S.cerevisiae. SEQ ID NOS 34 and 36 show the sequence ofthe two strands of the synthetic gene and the protein sequence is givenas SEQ ID NO 35. The method of Preparation 1 was followed for theconstruction of the ACT-2 gene except that the oligonucleotides differfrom those in preparation 1. FIG. 23 shows the annealed oligonucleotides used in the construction of the ACT-2 gene. The syntheticgene was cloned into plasmid pUC18 to create pUC18ACT-2.

Preparation 10--Construction of a Yeast Expression Vector for Human ACT2

The method of Preparation 2 was followed except that the ACT-2 gene frompUC18 ACT-2 was used in place of the pUC18-LD78. The resultant ACT-2expression vector was called pSW6 ACT-2.

Preparation 11--Expression of Human ACT-2 Synthetic Gene in Yeast

The method of Preparation 3 was followed except that pSW6 ACT-2 DNA wasa used as the expression vector.

Preparation 12--Purification of human ACT2 expressed from a syntheticgene in Yeast

Supernatant from the shake flasks described in Preparation 11 was spunat 6500 rpm for 15 minutes to clarify. Typically 3 liters of yeastsupernatant were adjusted to pH8 and 30 ml of Q-SEPAHROSE™pre-equilibrated in 50 mM Tris pH8.0 added. Protein was batch-adsorbedonto the resin overnight at 4° C. with gentle agitation. The resin wasallowed to settle and the supernatant removed. Resin was poured into acolumn 1.6 cm in diameter, washed with 10× volumes 50 mM Tris pH8.0,then eluted in 0.5M NaCl, 50 mM Tris pH8.0 (typically 50 ml totaleluent). The eluent was transferred into a prewetted SPECTRAPOR™dialysis membrane (3000 dalton cutoff) and dialysed against 10× volumesof 50 mM Tris pH8.0 at 4° C. with one change of buffer. The sample wasthen loaded onto an 8 ml Heparin-SEPHAROSE™ column (1.6 cm diameter)equilibrated in 50 mM Tris pH8.0 and the column washed with the samebuffer. ACT-2 was eluted in 50 mM Tris, 1M NaCl pH8.0. The eluent wasthen transferred to a prewetted SPECTRAPOR™ dialysis membrane (3000dalton cutoff) and dialysed against 10× volume 0.1% TFA at 4° C. withone change of buffer. After dialysis the sample was adjusted to 25%acetonitrile (final concentration). The protein sample was then loadedonto a 20 ml VYDAC™ C-18 (10μ pore-size) semi-preparative reverse phaseHPLC column pre-equilibrated at 3 ml/min in 20% acetonitrile, 0.1%Trifluoracetic acid (TFA) and eluted with a linear gradient from 20% to50% acetonitrile, 0.1% TFA over 40 minutes. Eluting fractions weredetected by u.v. absorbance at 280 nm and collected manually. The ACT-2protein was found to elute around 43% acetonitrile, 0.1% TFA. PurifiedACT-2 was freeze-dried and stored at -20° C.

Preparation 13--Confirmation of the Identity and Purity of Human LD78.Human ACT-2 and Murine MIP-1α expressed from Synthetic Genes in Yeast

A purity of greater than 97% was confirmed using a range of analyticalprocedures. Small aliquots (5 μg in 5 μl of sample buffer as describedin Comparative Example 2) of the dried material were analysed withsodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)using an 8-25% acrylamide gradient PHASTGELM™ (Pharmacia) using themanufacturers' recommended sample buffer and running programme. Gelswere stained and destained according to the manufacturers' recommendedprocedures.

Analytical reverse phase-HPLC was carried out using a 2 ml VYDAC™ C-18analytical reverse-phase column (5μ pore-size) equilibrated in 20%acetonitrile, 0.1% TFA at 1 ml/min. Approximately 10-50 μg of proteinwas loaded and eluted with a linear gradient of 20-50% acetonitrile,0.1% TFA at a flow rate of 1 ml/min over 20 minutes.

Near ultra-violet absorbance spectroscopy in the 240-320 nm range, witha 1 cm pathlength and 1 nm bandwidth was used to ensure that noturbidity (light scattering) arising from aggregated rSCI was present inthe purified preparation.

The identity of the purified proteins were confirmed from the N-terminalsequence and the mass of the product. Electrospray mass spectroscopy wascarried out on a VG BIO-Q™ spectrometer with 50 μg of sample dissolvedin 1:1 (v/v methanol/water containing 1% acetic acid. In some cases,substantial populations of LD78 protein+metal adduct mass peaks wereobserved. Typically potassium and copper were noted. Major coppercontamination was found to originate from a metal line within thespectrometer, however, the LD78 proteins were shown to tightly hold thismetal ion even in the large electric field applied on injection. Thestrength of association suggests that LD78 has an ability to bind thisdivalent metal ion with reasonable affinity if exposed to significantquantities.

N-terminal sequencing was carried out using an Applied Biosystems 471Asequenator, Applied Biosystems Ltd, Kelvin Close, Birchwood Science ParkNorth, Warrington WA3 7PB. Typically 250 picomoles of protein dissolvedin 0.1% TFA was loaded onto a BIOBRENE™ precycled glass fibre disc andsubjected to 14 cycles of automated Edman degradation. All proceduresand sequencing cycles were as recommended by the manufacturer.

COMPARATIVE EXAMPLES Comparative Example 1

Conformational Analysis of Recombinant Human LD78, Human ACT-2 andMurine MIP-1α

Near and far ultra-violet circular dichroism (u.v. c.d.) measurements ofMIP-1α, LD78 and ACT-2 were carried out using a Jobin-Yvon DichrographeVI. Samples were reconstituted in 10 mM acetic acid pH3.2 and a u.v.absorbance scan from 240-320 nm used to confirm the absence of proteinaggregates. The protein concentration was determined using calculatedvalues for the absorbance of a 1 mg/ml solution at 280 nm with a 1 cmpathlength of 1.37 for MIP-1α, 1.25 for LD78 and 1.57 for ACT-2. Meanresidue weights were calculated to be 114, 113.7 and 113.3 for MIP-1α,LD78 and ACT-2 respectively. Near u.v. c.d. spectra (250-320 nm) werecollected using a scan speed of 5 nm/min, 1 second response, 2 nmbandwidth and a 1 cm pathlength. Far u.v. c.d. spectra (190-250 nm) werecollected using a 10nm/min scan speed, 1 second response, 2 nm bandwidthand either a 0.01 cm or 0.05 cm pathlength. All spectra are calculatedand displayed as a mean residue molar ellipticity θ! with baselinesubtracted.

Analysis of the far ultra-violet circular dichroism of these sequencerelated proteins using the CONTIN™ programme (Provencher, Comput. Phys.Commun., 27, 229-242, (1982); Provencher & Gloeckner, Biochemistry, 20,33-37, (1981)) has confirmed that MIP-1α, LD78 and ACT-2 contain 14-18%helix and a high proportion of β-sheet structure which is consistentwith the known secondary structure contents of IL-8 (Clore et al.,J.Biol.Chem., 264, 18907-18911, (1989)) and PF-4 (St. Charles et al.,J.Biol.Chem., 264, 2092-2099, (1989)).

In the 250-320 nm wavelength range, circular dichroism spectra arisefrom disulphide bonds and aromatic groups such as tyrosine, tryptophanand to a lesser extent phenylalanine (Strickland, C.R.C.Crit.Rev.Biochem., 2, 113-175, (1974)). Circular dichroism bands in the near u.v.often (but not always) coincide with their chromophore absorption bands.The magnitude, sign (positive/negative) and wavelength position of thec.d. bands are highly sensitive to the conformational environment of thecontributing side-chain. Whilst no definitive set of rules exist forinterpretation of the contributions to near u.v. c.d. spectra, theintensity and position of shoulders, shape and maxima or minima cannevertheless be used to identify side-chain types. For example,characteristic tyrosine bands are often observed with minima centred at276 nm and 268 nm, in single tryptophan proteins the 0₋₋ 0¹ L_(b) bandis observed characteristically at 288-293 nm. Phenylalanine producesfine structure, often seen as shoulders in c.d. spectra, in the 250-270nm range. Disulphide bonds have very broad featureless contributionswhich are variable in intensity and can extend from 250 nm up to 360 nm.

Excepting Tyr3 of MIP-1α, the tyrosines and the single tryptophanresidues are conserved in the sequence of LD78 and MIP-1α. The near u.v.circular dichroism spectra of LD78 and MIP-1α are almost superimposable(FIG. 3). The intense negative ellipticity below 290 nm with the minimacentred at 268 nm and 276 nm is characteristic of tyrosine with somephenylalanine fine structure superimposed. The intensity of the spectrabetween 250-290 nm may reflect coupling of transitions between atyrosine residue and another aromatic group. The broad trough ofnegative ellipticity observed above 290 nm appears to exhibit a minimaat 296 nm with a broad trail to higher wavelength. This shape issomewhat unusual for a tryptophanyl band and it might, therefore,reflect a disulphide contribution. The data demonstrate that Tyr3 ofMIP-1α is not contributing to the spectrum. Given that the N-terminalregions of IL-8 and PF-4 are known to be disordered then the absence ofTyr3 c.d. in MIP-1α is not unexpected.

The near ultra-violet circular dichroism spectra demonstrate that theenvironment of aromatic amino acids in LD78 and MIP-1α is almostidentical. These data demonstrate that the two homologues have the sametertiary structure and conformation.

Comparison of the near u.v. c.d. of ACT-2 with MIP-1α highlightsdistinct differences in the shape and intensity of the spectra (FIG. 4).The ACT-2 spectrum shows a less intense negative tyrosine contributioncombined with a distinct 0₋₋ 0¹ L_(b) tryptophan contribution from the(conserved) single tryptophan residue-58. The only sequence differencebetween the proteins likely to contribute to the near u.v. c.d. is Tyr29in ACT-2 (Phe28 in MIP-1α). The nature of the shape and intensitychanges observed for the ACT-2 c.d. are not consistent with addition orcancellation simply of a tyrosine band. The data demonstrate, therefore,that there are distinct differences in the conformation of ACT-2compared to that of MIP-1α and LD78. These proteins all have similarmultimerisation properties as detailed in Comparative Example 3. Thevariation in conformation for ACT-2 is not therefore a result ofdifferent quaternary structure.

Comparative Example 2

LD78 is Immunologically Cross-Reactive with Anti-MIP-1α

5 μg each of MIP-1α, LD78, ACT-2 and human epidermal growth factor (as astandard marker) were dissolved in 5 μl of sample buffer (25 mM TrispH6.8, 2.3% sodium dodecyl sulphate, 5% β-mercaptoethanol, 10% glycerol,0.01% bromophenol blue) and heated at 90° C. for 5 minutes to reduce anddenature the protein. Approximately 1 μg of protein per track was loadedonto 2 identical 8-25% (acrylamide) SDS-PAGE PHASTGELS™; pre-stained lowmolecular weight markers (Bethesda Research Laboratories) were also runon each gel. The gels were electrophoresed using the manufacturers'recommended conditions. Following electrophoresis, one of the gels wasstained with 0.02% PHASTGEL BLUE R™, 30% methanol, 10% acetic acidfollowed by destaining in 30% methanol, 10% acetic acid. The second gelwas sandwiched between nitrocellulose membrane and electroblotted for 40minutes at 100 volts using 25 mM Tris, 192 mM glycine, 20% methanoltransfer buffer. After transfer of protein onto the nitrocellulosemembrane, the membrane was incubated in 0.5% casein, 154 mM NaCl, 20 mMTris pH7.4, 0.05% Triton blocking buffer for 1 hour at room temperature.The membrane was then incubated for an hour with a 1:5000 (v/v) dilutionof the primary antibody (polyclonal rabbit anti-MIP-1α, generated bystandard immunological techniques following immunisation with theprotein of Preparation 8) in blocking buffer at room temperature. Afterwashing 3×5 min with blocking buffer, the second antibody (anti-rabbitperoxidase conjugated (Sigma)) was incubated with the membrane at1:10000 (v/v) in blocking buffer for a further 1 hour at roomtemperature (Sigma Chemical Company Ltd, Fancy Road, Poole, Dorset BH177BR). After 3×5 min washes in 150 mM phosphate buffered saline pH7.4(PBS) the blot was developed in 25 ml of developing solution (0.04%3,3'-diaminobenzadine tetrahydrochloride, PBS, 0.015% cobalt chloride,0.015% ammonium nickel sulphate, 0.2% hydrogen peroxide). Developmentwas stopped by washing the membrane with distilled water. The membranewas then dried and photographed.

The immunoblot (FIG. 5; 8-25% SDS-PAGE (Reducing)) demonstrates thatthere is no cross-reaction of anti-MIP-1α with ACT-2, however, there isa strong cross-reaction with LD78. MIP-1α and LD78 have the same epitopeand immunological profile whereas ACT-2 is immunologically distinct.Together with the conformational data of Comparative Example 1, thisevidence strongly supports the suggestion that LD78 is the humanhomologue of murine MIP-1α.

Comparative Example 3

Characterization of the molecular weight of LD78 and MIP-1α (expressedfrom synthetic genes in yeast) under physiological conditions

LD78 and MIP-1α are non-glycosylated, with theoretical molecular weightsof 7712 Da and 7866 Da, respectively. ACT-2 has a theoretical molecularweight of 7704 Da, though the authentic molecule is thought to beglycosylated. Size exclusion chromatography (SEC) was carried out usinga SUPEROSE 12™ column attached to an Fast Protein Liquid Chromatographysystem (Pharmacia). The column was calibrated at 1 ml/min in 150 mMphosphate buffered saline, pH 7.4 (Sigma) using blue dextran, aldolase,bovine serum albumin, carbonic anhydrase and lysozyme as standards.Samples (50-100 μg) of MIP-1α, LD78 and ACT-2 were dissolved in 0.2 mlof 150 mM phosphate buffered saline, pH 7.4 (Sigma) and loaded onto thecolumn running at the calibrated speed of 1 ml/min. Eluting fractionswere detected by u.v. absorbance at 280 nm.

Reconstitution of each of lyophilized recombinant LD78, MIP-1α and ACT-2as described above yields a product which is predominantly a solublemultimeric complex when analysed by size exclusion chromatography (FIG.6). The soluble multimers range in size from 100,000 Da to >>200,000 Dawith the predominant weights apparently in the region of 350,000 Da. Thecolumn excludes particles of greater than 180,000 Da; therefore,accurate determinations of masses above this limit are impossible. Overa period of hours the multimeric complexes can form insoluble aggregateswhich visibly precipitate. A population of low molecular weight speciesis observed in the SEC profile of all three proteins. In view of theelution at >20,000 Da, and given that the SDS-PAGE results (described inComparative Example 2) show stable tetramers, it is suggested that theseproteins associate to stable tetramers similar to their sequencehomologue PF-4. The results described in detail in Comparative Examples4 and 5 confirm that a basic quaternary structural unit of thesemolecules is a tetramer.

Samples of LD78, MIP-1α and ACT-2 were also analysed using nativepolacrylamide gel electrophoresis. 5 μg of each protein werereconstituted in 25 mM Tris pH6.8, 10% glycerol, 0.01% bromophenol blue.Samples were loaded with high molecular weight markers (Flowgen) andhuman EGF standard and electrophoresed on a 5-50% GRADIPORE HYLINX™native gel (Flowgen) at 100 volts for 15 minutes in 0.0825M Tris,0.0808M boric acid, 0.003M EDTA, pH8.3. The gel was subsequentlyCoomassie blue-stained and destained (as described in ComparativeExample 2).

The stained gel (FIG. 7) shows that human EGF (6,200 daltons) standardruns at the correct weight under native conditions. MIP-1α, LD78 andACT-2 however, all run at the top of the gel with broad smearing bandsdemonstrating a range of molecular weight species. No low molecularweight species are observed.

The two techniques described above provide some quantitative estimatesof the molecular size of LD78 and MIP-1α in solution. In both cases,however, a solid support resin is present (acrylamide or SEPHADEX™)which can affect equilibrium populations of molecules. The recognizedmethod of absolute molecular mass determination in solution is bySedimentation Equilibrium in the analytical ultracentrifuge (see forexample Yphantis (1964) or Harding et al (1992)).

Using a BECKMAN OPTIMA™ XL-A analytical ultracentrifuge, protein solutedistributions can be recorded by u.v. absorbance during sedimentationequilibrium experiments (for example see Morgan et al, (1992)). In apopulation of protein molecules distributed at equilibrium through therotor cell, approximate values for the smallest (protein) mass insolution are obtained from the cell meniscus (M_(w) (ζ=0), and thelargest (protein) mass in solution determined from the cell base (M_(w)(ζ=1). This technique provides the whole-cell weight average molecularweight (M.sup.∘_(w)) (i.e. the average molecular weight of solutedistributed through the rotor cell). In this manner the self-associationproperties (if present) of a protein molecule can accurately bedetermined from the measured polydispersity in the observed mass ranges.Characteristically, such polydisperse solutions show an upward curvaturewhen logarithm of absorbance is plotted as a function of the normalisedradial displacement parameter (ζ) in analyses of the type detailed byCreeth & Harding (J.Biochem. Biophys.Methods, 7 25-34 (1982)) and Creeth& Pain, (Prog. Biophys & Mol. Biol. 17 217-287 (1967)). Proteins whichexist in solution at a single defined mass (i.e. a monodispersepopulation) exhibit linear plots of Ln A vs ζ. Non-ideal solutionconditions typically yield a downward curvature of Ln A vs ζ. It ispossible for the effects of polydispersity and nonideality to canceleach other and give a linear plot of Ln A vs ζ (Creeth & Pain, loc. cit.(1967)). This must be considered during data interpretation.

The sedimentation equilibrium behaviour of pure wild type LD78 wasmeasured at 20° C. with a protein concentration of 0.5 mg/ml using theOPTIMA™ XL-A ultracentrifuge with a rotor speed of 9000, 10000 or 12000r.p.m. and absorbance detection at 278 nm. For masses in the range ofmonomers, a rotor speed of 28000 r.p.m. is necessary at 20° C. Themethodology and analysis were as described by Morgan et al, (1992). Theresults (FIG. 8) showed that wild type LD78 exists in solution as apolydisperse population of protein species ranging in mass from approx.10,000 Da (M_(w) (ζ=0)) to 250,000 Da (M_(w) (ζ=1)). The whole cellweight average molecular weight (M.sup.∘_(w)) was found to be 160,000Da.

Pure MIP-1α was analysed in the same manner except that the rotor speedwas 15,000 r.p.m. In this case, the protein was shown to exist as apolydisperse solution of protein species ranging in mass from 230,000 Da(M_(w) (ζ=0)) to 350,000 Da (M_(w) (ζ=1)) with (M.sup.∘_(w))=310,000 Da.

The results from the independent techniques described above confirm thatMIP-1α, LD78 and ACT-2 form large, soluble, heterogenous, multimericcomplexes on reconstitution in low ionic strength aqueous buffers.

It is known that 0.5M NaCl prevents formation of the high molecularweight forms of MIP-1α and that in culture medium, around 5% of thetotal protein is a low molecular weight form (Oh et al.(1991)). Ourstudies demonstrate that in the absence of salt (i.e. in native PAGEsample buffer) no low molecular weight forms are present. Inphysiological ionic strength (150 mM phosphate buffered saline, pH7.4),a distinct population of low molecular weight protein species is presentas seen in the size exclusion profiles. An equilibrium is, therefore,present between the high and low molecular weight species. Thisequilibrium is influenced by the ionic strength of the buffer--seeComparative Example 4.

Comparative Example 4

Characterization of the molecular weight of LD78 and MIP-1α (expressedfrom synthetic genes in yeast) in 10 mM MES, 500 mM NaCl pH6.5

Salt concentrations of 0.5M have been claimed to prevent formation ofhigh molecular weight forms of MIP-1α (Wolpe and Cerami (1989). In orderto characterize fully the effect of salt and to elucidate theassociation pathway of SCI multimers, the molecular weight of LD78 andMIP-1α were examined in conditions of high ionic strength.

Size exclusion chromatography was carried out using a SUPEROSE 12™column attached to an FPLC system (Pharmacia). The column was calibratedat 1 ml/min in 10 mM MES (Sigma), 500 mM NaCl pH6.5 using the standardsdescribed in Comparative Example 3. Samples (100 μg) of MIP-1α and LD78were dissolved in 0.2 ml of 10 mM MES (Sigma), 500 mM NaCl pH6.5 andloaded onto the column running at the calibrated speed of 1 ml/min.Eluted fractions were detected by u.v. absorbance at 280 nm.

Reconstitution of the lyophilized recombinant LD78 under theseconditions gives an SEC elution profile containing a single symmetricalpeak of mass around 20-25 KDa. The peak symmetry indicates that a singlehomogenous population of protein molecules exists. It is unclear fromthe determined mass whether trimeric or tetrameric LD78 represents theobserved species.

Reconstitution of lyophilized recombinant MIP-1α under these conditionsgives an SEC elution profile containing an asymmetric peak ofapproximate mass 25 kDa trailing down to around 5 kDa. The shape of thepeak suggests the protein exists in a number of mass species under theseconditions. The elution profile most probably reflects the presence oftetramer, dimer and monomer populations in solution.

Analytical ultracentrifugation of LD78 was carried out in these bufferconditions as described in Comparative Example 3. The whole cell weightaverage molecular weight was calculated to be a single population withmolecular mass of 29 ±2 kDa. Under these conditions of 10 mM MES, 500 mMNaCl pH6.4, LD78 exists in solution as a defined tetramer in the absenceof higher and lower molecular weight forms (FIG. 8).

These data demonstrate that ionic interactions play a key role in theassociation of LD78 tetramers to form the large heterogeneous multimers.

Comparative Example 5

Characterization of the molecular weight of LD78 (expressed from asynthetic gene in yeast) in 50 mM Tris, 1M Glycine pH 8.3

In order to correllate the molecular mass profiles obtained on nativePAGE immunoblots described in Comparative Example 3 with data generatedfrom other methods of mass determination, SEC was carried out in nativePAGE buffer. SUPERDEX 75™ FPLC resin has a comparable mass resolutionrange (3-70 kDa) as the Biorad (MINIPROTEAN™ 12% acrylamide) pre-castgels. This size exclusion column was, therefore, used to recreate thegel conditions as closely as possible. 100 μg of LD78 was reconstitutedin 0.2 ml of 50 mM Tris, 1M Glycine pH8.3 buffer and injected at a flowrate of 1 ml/min onto the SUPERDEX 75™ column equilibrated in the samebuffer. Eluting fractions were detected by u.v. absorbance at 280 nm.

The SEC elution profile of LD78 shows a major asymmetric peak of highmolecular weight (>70 kDa) protein partially excluded from the column, asmall dimer peak at approx. 15 KDa and a major symmetrical peakcorresponding to the monomer mass around 8,000 Da. The presence of alarge population of monomeric LD78 in equilibrium with high molecularweight multimers suggests that the quaternary structure of LD78 isextremely sensitive either to:

(i) a 0.9 unit shift in pH between this buffer system and that inComparative Example 3 or

(ii) the presence of significant concentrations of the free amino acidglycine.

Sedimentation equilibrium studies of LD78 (method as described inComparative Example 3) at a protein concentration of 0.5 mg/ml underthese buffer conditions reveal the presence of a polydisperse populationof mass species ranging from 8,000 Da (M_(w) (ζ=0)) to >300,000 Da(M_(w) (ζ=1)).

As described in Preparation 13, electrospray mass spectroscopy revealsthe presence of mono-valent and di-valent metal ions bound to purifiedLD78. Many chemical methods involve the use of metal ions to chelate tofree amino and carboxyl groups of amino acids to enable the modificationof side-chain groups in reaction mixtures (Chemistry of the Amino Acids,Volume 1--Chapter 6, Krieger Publishing Florida, ed. Greenstein & Winitz(1961)). The glycine present in this buffer could therefore act as achelator for metal ions. Only a small population of dimeric LD78 isevident in the SEC profile and no tetrameric species are observed. It issuggested, therefore, that metal ions can play a role in thestabilization of both the LD78 tetramer and dimer units.

Comparative Example 6

Characterization of the molecular weight of LD78 (expressed from asynthetic gene in yeast) in 10 mM acetic acid pH3.2

The stem cell inhibitor protein LD78 is very soluble in mild acidicconditions. Size exclusion chromatography is not ideal under acidicconditions, therefore, analytical ultracentrifugation of LD78 wascarried out. At a protein concentration of 0.5 mg/ml in 10 mM aceticacid pH3.2, a monodisperse mass species of 33±3 kDa was observed (FIG.8). This mass equates to a monodisperse population of tetramers.

The relatively low ionic strength acidic conditions may titrate Gluand/or Asp groups involved in the electrostatic interactions involved inthe association of tetramers to multimers described in ComparativeExample 4.

Comparative Example 7

Spectroscopic assay for the state of multimerisation of LD78 proteinexpressed from a synthetic gene in yeast

LD78 exists in solution as a tetramer in 10 mM acetic acid pH3.2 and 10mM MES, 500 mM NaCl pH6.4 (Comparative Example 4 & 6). In solutions suchas 150 mM PBS pH7.4 the protein is present as a heterogeneous populationof soluble multimeric complexes stabilized by electrostatic interactionsbetween charged side-chains (Comparative Example 3).

The near u.v. c.d. spectra of LD78 (measured as described in ComparativeExample 1) in 10 mM acetic acid pH3.2 and 10 mM MES, 500 mM NaCl pH6.4are identical (FIG. 9). This data demonstrates that the bufferconditions used to produce tetrameric LD78 do not affect the tertiaryconformation of the protein.

Comparison of the near u.v. c.d. spectrum of tetrameric LD78 with thatof the high molecular weight multimers in 150 mM PBS pH7.4 (FIG. 10)shows that the asymmetric environment of Trp57 is different in the twostates (See Comparative Example 1 for discussion of c.d. spectrainterpretation).

Examination of the steady state fluorescence emission spectra (method asdescribed in Comparative Example 1) of LD78 in 10 mM acetic acid, 10 mMMES, 500 mM NaCl pH6.4 and 150 mM PBS pH7.4 (FIG. 11) shows that in thehigh molecular weight multimers, quenching of emission intensity occurs.The fact that λ_(max) has not shifted, indicates that no conformationalchanges have occurred giving rise to quenching. It is known thatelectrostatic interactions proximal to tryptophan residues will quenchthe emission of fluorescence energy (Lackowicz (1983)). Stabilizinginteractions between acidic and basic amino acids are known to be keyfor the association of tetramers to form multimeric complexes(Comparative Example 4). The change in environment observed for Trp-57is entirely consistent with the formation of an ionic interactionspatially close to the side-chain.

Near u.v. c.d. and/or fluorescence emission spectroscopy can provide asensitive probe for the multimer state of LD78.

EXAMPLES Example 1

Design and Construction of LD78 Variant Gln48>Glu (Mutant 1) andConstruction of an LD78 Gln48>Glu Expression Vector

The strategy for construction of an LD78 variant (eg. Gln48>Glu) byoligonucleotide directed mutagenesis and molecular cloning is describedbelow. Mutagenesis was carried out according to the method of Kunkel etal., Methods in Enzymology 154 367-382 (1987). Host strains and methodsare described below.

E. coli strains

RZ1032 is a derivative of E. coli that lacks two enzymes of DNAmetabolism: (a) dUTPase (dut), the lack of which results in a highconcentration of intracellular dUTP, and (b) uracil N-glycosylase (ung)which is responsible for removing mis-incorporated uracils from DNA(Kunkel et al., loc. cit.). A suitable alternative strain is CJ236,available from Bio-Rad Laboratories, Watford WD1 8RP, United Kingdom.The principal benefit is that these mutations lead to a higher frequencyof mutants in site directed mutagenesis. RZ1032 has the followinggenotype:

HfrKL16PO/45 lysA961-62), dut1, ung1, thi1, recA, Zbd-279::Tn10, supE44

JM103 is a standard recipient strain for manipulations involving M13based vectors. The genotype of JM103 is Δ (lac-pro), thi, supE, strA,endA, sbcB15, hspR4, F' traD36, proAB, lacIq, lacZΔM15. A suitablecommercially available alternative E. coli strain is E. coli JM109,available from Northumbria Biologicals Ltd.

Mutagenesis

Prior to mutagenesis it was necessary to transfer the LD78 gene into asuitable vector. This was accomplished as described below.

pSW6 LD78 plasmid DNA of Preparation 2 was prepared and an aliquot wastreated with restriction enzymes HindIII and BamHI. A ca. 235 bp DNAfragment from this digestion was gel purified and ligated to HindIII andBamHI treated E. coli bacteriophage M13 mp19 DNA. The products of theligation reaction were transfected into competent cells of E. colistrain JM103. Single stranded DNAs from 6 putative recombinant plaqueswere then prepared and sequenced, by the dideoxy method, with auniversal primer BB22 (5'-GTTTTCCCAGTCACGAC-3' (SEQ ID NO 47)).

Single stranded DNA of M13mp19-LD78 was prepared from E. coli RZ1032 andused as a template for oligonucleotide directed mutagenesis as describedby Kunkel et al., using 22-mer oligonucleotide BB6298(5'-GCACAGACTTCTCTCGAGCGCT-3' (SEQ ID NO 48)). The required mutant(pGHC600) was identified by dideoxy sequence analysis of single strandedDNAs prepared from putative mutant plaques. Primer BB22 (see above) wasused as the sequencing primer in all cases. Double stranded replicativeform (RF) DNA was prepared from the bacteriophage carrying the requiredmutation. The RF DNA was then digested with HindIII and BamHI. The DNAfragment carrying the LD78 Gln48>Glu gene was then purified afterelectrophoretic separation on a low gelling temperature agarose gel bystandard methods. This fragment was then ligated to HindIII and BamHItreated pSW6 DNA to create an expression vector for the LD78 Gln48>Glugene. The sequence of a correct clone (pSJE50) was verified by plasmidDNA sequencing. Expression of the mutant LD78 protein was achievedaccording to methods described in Preparation 3.

Example 2

Design and Construction of LD78 Variant Lys44>Glu Arg45>Gln (Mutant 2)and Construction of an LD78 Lys44>Glu Arg45>Gln Expression Vector

LD78 Lys44>Glu Arg45>Gln was constructed and cloned into the pSW6 yeastexpression vector as described in Example 1. A 30-mer oligonucleotideBB6299 (5'-GACTTGTCTCGATTGCTCAGTCAAGAAGAT-3' (SEQ ID NO 49)) was used tomutate the LD78 gene and a correct clone identified (pGHC601). Themutant gene was cloned into the expression vector to create pSJE5 1.Expression of the mutant LD78 protein was achieved according to methodsdescribed in Preparation 3.

Example 3

Design and Construction of LD78 Variant Ala9>Ser (Mutant 3) andConstruction of an LD78 Ala9>Ser Expression Vector

LD78 Ala9>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 22-mer oligonucleotide BB6300(5'AAACAACAAGAGGTTGGAGTGT-3' (SEQ ID NO 50)) was used to mutate the LD78gene and a correct clone identified (pGHC602). The mutant gene wascloned into the expression vector to create pSJE52. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 4

Design and Construction of LD78 Variant Phe28>Ser (Mutant 4) andConstruction of an LD78 Phe28>Ser Expression Vector

LD78 Phe28>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 25-mer oligonucleotide BB6381(5'-GAAGAAGTITCA(G/C/T)AGTAGTCAGCAA-3' SEQ ID NO 51) was used to mutatethe LD78 gene and a correct clone identified (pGHC603). The mutant genewas cloned into the expression vector to create pSJE53. Expression ofthe mutant LD78 protein was achieved according to methods described inPreparation 3.

Example 5

Design and Construction of LD78 Variant Arg17>Ser (Mutant 5) andConstruction of an LD78 Arg 17>Ser Expression Vector

LD78 Arg17>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 25-mer oligonucleotide BB6302(5'-GTGGAATTTGAGAAGAGGTGTAAGA-3' SEQ ID NO 52) was used to mutate theLD78 gene and a correct clone identified (pGHC604). The mutant gene wascloned into the expression vector to create pSJE54. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 6

Design and Construction of LD78 Variant Phe23>Asn Ile24>Thr (Mutant 6)and Construction of an LD78 Phe23>Asn Ile24>Thr Expression Vector

LD78 Phe23>Asn Ile24>Thr was constructed and cloned into the pSW6 yeastexpression vector as described in Example 1. A 27-mer oligonucleotideBB6303 (5'-GTAGTCAGCAGTGTTATTTTGTGGAAT-3' SEQ ID NO 53) was used tomutate the LD78 gene and a correct clone identified (pGHC605). Themutant gene was cloned into the expression vector to create pSJE55.Expression of the mutant LD78 protein was achieved according to methodsdescribed in Prepapation 3.

Example 7

Design and Construction of LD78 Variant Asp26>Ala (Mutant 10) andConstruction of an LD78 Asp26>Ala Expression Vector

LD78 Asp26>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 25-mer oligonucleotide BB6625(5'-TTTCAAAGTAG(G/A)CAGCAATGAAATT-3' SEQ ID NO 54) was used to mutatethe LD78 gene and a correct clone identified (pGHC609). The mutant genewas cloned into the expression vector to create pSJE59. Expression ofthe mutant LD78 protein was achieved according to methods described inPrepapation 3.

Example 8

Design and Construction of LD78 Variant Phe12>Gln (Mutant 11) andConstruction of an LD78 Phe12>Gln Expression Vector

LD78 Phe12>Gln was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 24-mer oligonucleotide BB6301(5'-AGGTGTAAGATTGACAACAAGCGG-3' SEQ ID NO 55) was used to mutate theLD78 gene and a correct clone identified (pGHC610). The mutant gene wascloned into the expression vector to create pSJE60. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 9

Design and Construction of LD78 Variant Ile24>Thr (Mutant 13) andConstruction of an LD78 Ile24>Thr Expression Vector

LD78 Ile24>Thr was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 25-mer oligonucleotide BB6382(5'-AGTAGTCAGCA(G/C/T)TGAAATTTTGTGG-3' SEQ ID NO 56) was used to mutatethe LD78 gene and a correct clone identified (pGHC612). The mutant genewas cloned into the expression vector to create pSJE62. Expression ofthe mutant LD78 protein was achieved according to methods described inPreparation 3.

Example 10

Design and Construction of LD78 Variant Ile40>Arg (Mutant 14) andConstruction of an LD78 Ile40>Arg Expression Vector

LD78 Ile40>Arg was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 24-mer oligonucleotide BB6383(5'-TAGTCAAGAATCTGACACCTGGCT-3' SEQ ID NO 57) was used to mutate theLD78 gene and a correct clone identified (pGHC613). The mutant gene wascloned into the expression vector to create pSJE63. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 11

Design and Construction of LD78 Variant Arg47>Glu (Mutant 15) andConstruction of an LD78 Arg47>Glu Expression Vector

LD78 Arg47>Glu was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 26-mer oligonucleotide BB6384(5'-GCACAGACTTGTTCCGAGCGCTTAGT-3' SEQ ID NO 58) was used to mutate theLD78 gene and a correct clone identified (pGHC614). The mutant gene wascloned into the expression vector to create pSJE64. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 12

Design and Construction of LD78 Variant Lys60>Gln Asp64>Asn (Mutant 16)and Construction of an LD78 Lys60>Gin Asp64>Asn Expression Vector

LD78 Lys60>Gln Asp64>Asn was constructed and cloned into the pSW6 yeastexpression vector as described in Example 1. A 35-mer oligonucleotideBB6385 (5'-AATTCCAAGTTAGAAACATATTGTTGAACCCATTC-3' SEQ ID NO 59) was usedto mutate the LD78 gene and a correct clone identified (pGHC615). Themutant gene was cloned into the expression vector to create pSJE65.Expression of the mutant LD78 protein was achieved according to methodsdescribed in Preparation 3.

Example 13

Design and Construction of LD78 Variant Phe28>Glu (Mutant 17) andConstruction of an LD78 Phe28>Glu Expression Vector

LD78 Phe28>Glu was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 25-mer oligonucleotide BB6345(5'-GAAGAAGTTTCTTCGTAGTCAGCAA-3' SEQ ID NO 60) was used to mutate theLD78 gene and a correct clone identified (pGHC616). The mutant gene wascloned into the expression vector to create pSJE66. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 14

Design and Construction of LD78 Variant Ile24>Asn (Mutant 24) andConstruction of an LD78 Ile24>Asn Expression Vector

LD78 Ile24>Asn was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 25-mer oligonucleotide BB6382(5'-AGTAGTCAGCA(G/C/T)TGAAATTTTGTGG-3' SEQ ID NO 56) was used to mutatethe LD78 gene and a correct clone identified (pGHC623). The mutant genewas cloned into the expression vector to create pSJE73. Expression ofthe mutant LD78 protein was achieved according to methods described inPreparation 3.

Example 15

Design and Construction of LD78 Variant Phe28>Glu Gln48>Glu (Mutant 25)and Construction of an LD78 Phe28>Glu Gln48>Glu Expression Vector

LD78 Phe28>Glu Gln48>Glu was constructed and cloned into the pSW6 yeastexpression vector essentially as described in Example 1. A 27-meroligonucleotide BB7015 (5'-TGAGAAGAAGTTTCTTCGTAGTCAGCA-3' SEQ ID NO 61)was used to mutate the LD78 Gln48>Glu gene (pGHC600 of Example 1) and acorrect clone identified (pGHC624). The mutant gene was cloned into theexpression vector to create pSJE74. Expression of the mutant LD78protein was achieved according to methods described in Preparation 3.

Example 16

Design and Construction of LD78 Variant Phe28>Glu Arg47>Glu (Mutant 26)and Construction of an LD78 Phe28>Glu Arg47>Glu Expression Vector

LD78 Phe28>Glu Arg47>Glu was constructed and cloned into the pSW6 yeastexpression vector essentially as described in Example 1. A 27-meroligonucleotide BB7015 (5'-TGAGAAGAAGTTTCTTCGTAGTCAGCA-3' SEQ ID NO 61)was used to mutate the LD78 Arg47>Glu gene (pGHC614 of Example 11) and acorrect clone identified (pGHC625). The mutant gene was cloned into theexpression vector to create pSJE75. Expression of the mutant LD78protein was achieved according to methods described in Preparation 3.

Example 17

Design and Construction of LD78 Variant Glu55>Arg Glu56>Arg (Mutant 27)and Construction of an LD78 Glu55>Arg Glu56>Arg Expression Vector

LD78 Glu55>Arg Glu56>Arg was constructed and cloned into the pSW6 yeastexpression vector as described in Example 1. A 27-mer oligonucleotideBB9112 (5'-TTGAACCCAGCGGCGAGATGGGTCAGC-3' SEQ ID NO 62) was used tomutate the LD78 gene and a correct clone identified (pGHC626). Themutant gene was cloned into the expression vector to create pSJE76.Expression of the mutant LD78 protein was achieved according to methodsdescribed in Preparation 3.

Example 18

Design and Construction of LD78 Variant Glu29>Arg (Mutant 28) andConstruction of an LD78 Glu29>Arg Expression Vector

LD78 Glu29>Arg was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 24-mer oligonucleotide BB9109(5'-TTGAGAAGAAGTTCTAAAGTAGTC-3' SEQ ID NO 63) was used to mutate theLD78 gene and a correct clone identified (pGHC627). The mutant gene wascloned into the expression vector to create pSJE77. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 19

Design and Construction of LD78 Variant Gln 18>Glu (Mutant 29) andConstruction of an LD78 Gln 18>Glu Expression Vector

LD78 Gln18>Glu was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 24-mer oligonucleotide BB9110(5'-ATTTTGTGGAATITCTCTAGAGGT-3' SEQ ID NO 64) was used to mutate theLD78 gene and a correct clone identified (pGHC628). The mutant gene wascloned into the expression vector to create pSJE78. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 20

Design and Construction of LD78 Variant Arg17>Ser Gln18>Glu (Mutant 30)and Construction of an LD78 Arg 17>Ser Gln18>Glu Expression Vector

LD78 Arg17>Ser Gln18>Glu was constructed and cloned into the pSW6 yeastexpression vector as described in Example 1. A 30-mer oligonucleotideBB9111 (5'-ATTTTGTGGAATTMCAGAAGAGGTGTAAGA-3' SEQ ID NO 65) was used tomutate the LD78 gene and a correct clone identified (pGHC629). Themutant gene was cloned into the expression vector to create pSJE79.Expression of the mutant LD78 protein was achieved according to methodsdescribed in Preparation 3.

Example 21

Design and Construction of LD78 Variant Ser-Ala-LD78 (Mutant 31) andConstruction of a Ser-Ala-LD78 Expression Vector

Ser-Ala-LD78 was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 30-mer oligonucleotide BB9104(5'-AGCAGCCAAGGAAGCAGATCTTTTATCCAA-3' SEQ ID NO 66) was used to mutatethe LD78 gene and a correct clone identified (pGHC630). The mutant genewas cloned into the expression vector to create pSJE80. Expression ofthe mutant LD78 protein was achieved according to methods described inPreparation 3.

Example 22

Design and Construction of LD78 Variant Leu-Ser-Ala-Ser1>Pro LD78(Mutant 32) and Construction of a Leu-Ser-Ala-Ser1>Pro LD78 ExpressionVector

Leu-Ser-Ala-Ser1>Pro LD78 (in which the residues Leu, Ser and Ala havebeen added to the N-terminus of LD78 and in which Pro has beensubstituted for Ser1) was constructed and cloned into the pSW6 yeastexpression vector as described in Example 1. A 36-mer oligonucleotideBB9105 (5'-GTCAGCAGCCAATGGAGCAGACAATCTTTTATCCAA-3' SEQ ID NO 67) wasused to mutate the LD78 gene and a correct clone identified (pGHC631).The mutant gene was cloned into the expression vector to create pSJE81.Expression of the mutant LD78 protein was achieved according to methodsdescribed in Preparation 3.

Example 23

Design and Construction of an LD78 Variant With the First ThreeN-terminal Amino Acids Deleted (N1-3 LD78) (Mutant 33) and Constructionof an N1-3 LD78 Expression Vector

N1-3 LD78 was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 24-mer oligonucleotide BB9106(5'-TGGAGTGTCAGCTCTTTATCCAA-3' SEQ ID NO 68) was used to mutate the LD78gene and a correct clone identified (pGHC632). The mutant gene wascloned into the expression vector to create pSJE82. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 24

Design and Construction of LD78 Variant Ala-Ser1>Pro LD78 (Mutant 34)and Construction of an Ala-Ser1>Pro LD78 Expression Vector

Ala-Ser1>Pro LD78 (in which the residue Ala has been added to theN-terminus of LD78 and in which Pro has been substituted for Ser1) wasconstructed and cloned into the pSW6 yeast expression vector asdescribed in Example 1. A 30-mer oligonucleofide BB9103(5'-GTCAGCAGCCAATGGAGCTCTTTTATCCAA-3' SEQ ID NO 69) was used to mutatethe LD78 gene and a correct clone identified (pGHC633). The mutant genewas cloned into the expression vector to create pSJE83. Expression ofthe mutant LD78 protein was achieved according to methods described inPreparation 3.

Example 25

Design and Construction of LD78 Variant Leu-Ser-Ala-Ser1>Pro Gly38>SerSer46>Gly (Mutant 35) and Construction of a Leu-Ser-Ala-Ser1>ProGly38>Ser Ser46>Gly Expression Vector

LD78 Leu-Ser-Ala-Ser1>Pro Gly38>Ser Ser46>Gly (in which the residuesLeu, Ser and Ala have been added to the N-terminus of LD78 and in whichPro has been substituted for Ser1, Ser has been substituted for Gly 38and Gly has been substituted for Ser 46) was constructed and cloned intothe pSW6 yeast expression vector essentially as described in Example 1.A 48-mer oligonucleotide BB9108(5'-ACAGACTTGTCTACCGCGCTTAGTCAAGAAGATGACAGATGGCTTGGA-3' SEQ ID NO 70)was used to mutate the Leu-Ser-Ala-Ser1>Pro LD78 gene (pGHC631 ofExample 22) and a correct clone identified (pGHC634). The mutant genewas cloned into the expression vector to create pSJE84. Expression ofthe mutant LD78 protein was achieved according to methods described inPreparation 3.

Example 26

Design and Construction of an LD78 Variant With the First ThreeN-terminal Amino Acids Deleted (N1-3) and Thr15>Phe (Mutant 36) andConstruction of an (N1-3) Thr15>Phe LD78 Expression Vector

N1-3 Thr15>Phe LD78 was constructed and cloned into the pSW6 yeastexpression vector essentially as described in Example 1. A 24-meroligonucleotide BB9107 (5'-AATTTGTCTAGAGAAGTAAGAGAA-3' SEQ ID NO 71) wasused to mutate the N1-3 LD78 gene (pGHC632 of Example 23) and a correctclone identified (pGHC635). The mutant gene was cloned into theexpression vector to create pSJE85. Expression of the mutant LD78protein was achieved according to methods described in Preparation 3.

Example 27

Design and Construction of LD78 Variant Gln48>Ser (Mutant 70) andConstruction of an LD78 Gln48>Ser Expression Vector

LD78 Gln48>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 21-mer oligonucleotide BB9512(5'-CAGCACAGACAGATCTCGAGC-3' SEQ ID NO 72) was used to mutate the LD78gene and a correct clone identified (pGHC670). The mutant gene wascloned into the expression vector to create pRC59/70. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 28

Design and Construction of LD78 Variant Asp26>Ser (Mutant 39) andConstruction of an LD78 Asp26>Ser Expression Vector

LD78 Asp26>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. An 18-mer oligonucleotide BB9432(5'-CAAAGTAGGAAGCAATGA-3' SEQ ID NO 73) was used to mutate the LD78 geneand a correct clone identified (pGHC638). The mutant gene was clonedinto the expression vector to create pSJE88. Expression of the mutantLD78 protein was achieved according to methods described in Preparation3.

Example 29

Design and Construction of LD78 Variant Phe12>Ala (Mutant 77) andConstruction of an LD78 Phe12>Ala Expression Vector

LD78 Phe12>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide BB9519(5'-GTGTAAGAGGCACAACAAG-3' SEQ ID NO 74) was used to mutate the LD78gene and a correct clone identified (pGHC676). The mutant gene wascloned into the expression vector to create pDB127. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 30

Design and Construction of LD78 Variant LD78 Phe28>Ala (Mutant 85) andConstruction of an LD78 Phe28>Ala Expression Vector

LD78 Phe28>Ala was constructed and is cloned into the pSW6 yeastexpression vector as described in Example 1. A 19-mer oligonucleotideBB9527 (5'-GAAGTTTCAGCGTAGTCAG-3' SEQ ID NO 75) was used to mutate theLD78 gene and a correct clone identified (pGHC684). The mutant gene wascloned into the expression vector to create pDB130. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 31

Design and Construction of LD78 Variant Ile24>Ala (Mutant 38) andConstruction of an LD78 Ile24>Ala Expression Vector

LD78 Ile24>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 21-mer oligonucleotide BB9431(5'-GTAGTCAGCAGCGAAATTTTG-3' SEQ ID NO 76) was used to mutate the LD78gene and a correct clone identified (pGHC637). The mutant gene wascloned into the expression vector to create pSJE87. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 32

Design and Construction of LD78 Variant Ile40>Ala (Mutant 92) andConstruction of an LD78 Ile40>Ala Expression Vector

LD78 Ile40>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide, BB9534(5'-GTCAAGAAGGCGACACCTG-3' SEQ ID NO 77) was used to mutate the LD78gene and a correct clone identified (M13DB104). The mutant gene wascloned into the expression vector to create pDB114. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 33

Design and Construction of LD78 Variant LD78 Arg47>Ser (Mutant 44) andConstruction of an LD78 Arg47>Ser Expression Vector

LD78 Arg47>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 21-mer oligonucleotide BB9437(5'-CACAGACTTGAGACGAGCGCT-3' SEQ ID NO 78) was used to mutate the LD78gene and a correct clone identified (pGHC643). The mutant gene wascloned into the expression vector to create pDB144. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 34

Design and Construction of LD78 Variant Glu29>Ser (Mutant 40) andConstruction of an LD78 Glu29>Ser Expression Vector

LD78 Glu29>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 22-mer oligonucleotide BB9433(5'-GAGAAGAAGTAGAAAAGTAGTC-3' SEQ ID NO 79) was used to mutate the LD78gene and a correct clone identified (pGHC639). The mutant gene wascloned into the expression vector to create pDB135. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 35

Design and Construction of LD78 Variant Gln 18>Ser (Mutant 64) andConstruction of an LD78 Gln18>Ser Expression Vector

LD78 Gln 18>Ser was constructed and cloned into the pSW6 yeastexpression vector as described in Example 1. A 21-mer oligonucleotideBB9506 (5'-TTGTGGAATAGATCTAGAGG-3' SEQ ID NO 80) was used to mutate theLD78 gene and a correct clone identified (pGHC663). The mutant gene wascloned into the expression vector to create pRC59/64. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 36

Design and Construction of LD78 Variant Asp5>Arg (Mutant 104) andConstruction of an LD78 Asp5>Arg Expression Vector

LD78 Asp5>Arg was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 23-mer oligonucleotide BB10194(5'-GGTTGGAGTGCGAGCAGCCAAGG-3' SEQ ID NO 81) was used to mutate the LD78gene and a correct clone identified (pGHC566). The mutant gene is clonedinto the expression vector according to the method of Example 1 andexpression of the mutant LD78 protein is achieved according to methodsdescribed in Preparation 3.

Example 37

Construction of LD78 Variant Arg17>Glu, (Gln, Ile, Pro) Insertionbetween residues 20 and 21 (Mutant 105) and Construction of an LD78Arg17>Glu (Gln, Ile, Pro) Insertion between residues 20 and 21Expression Vector

LD78 Arg17>Glu (Gln, Ile, Pro) Insertion between residues 20 and 21 isconstructed and cloned into the pSW6 yeast expression vector asdescribed in Example 1. A 22-mer oligonucleotide BB10195(5'-GGAATTTGTTCAGAGGTGTAAG-3' SEQ ID NO 82) was used to mutate the LD78gene and a clone containing the desired site-directed sequence mutationand an additional unintentional three amino-acid insertion identified(M13DB120). The mutant gene was cloned into the expression vector tocreate pDB138. Expression of the mutant LD78 protein was achievedaccording to methods described in Preparation 3.

Example 38

Design and Construction of LD78 Variant Ser46>Glu (Mutant 106) andConstruction of an LD78 Ser46>Glu Expression Vector

LD78 Ser46>Glu is constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 27-mer oligonucleotide BB10196(5'-GCACAGACTTGTCMTlCGCGCTTAGTC-3' SEQ ID NO 83) was used to mutate theLD78 gene and a correct clone was identified (M13DB121). The mutant genewas cloned into the expression vector to create pDB146. Expression ofthe mutant LD78 protein was achieved according to methods described inPreparation 3.

Example 39

Design and Construction of LD78 Variant Leu2>Glu (Mutant 107) andConstruction of an LD78 Leu2>Glu Expression Vector

LD78 Leu2>Glu is constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 29-mer oligonucleotide BB10197(5'-GGAGTGTCAGCAGCTTCGGATCTTTATC-3' SEQ ID NO 84) was used to mutate theLD78 gene and a correct clone identified (M13DB122). The mutant gene wascloned into the expression vector to create pDB139. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 40

Design and Construction of LD78 Variant Ala3>Glu (Mutant 108) andConstruction of an LD78 Ala3>Glu Expression Vector

LD78 Ala3>Glu is constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 22-mer oligonucleotide BB10198(5'-GGAGTGTCAGCTTCCAAGGATC-3' SEQ ID NO 85) was used to mutate the LD78gene and a correct clone identified (M13DB123). The mutant gene iscloned into the expression vector according to the method of Example 1and expression of the mutant LD78 protein is achieved according tomethods described in Preparation 3.

Example 41

Design and Construction of LD78 Variant Ala4>Glu (Mutant 109) andConstruction of an LD78 Ala4>Glu Expression Vector

LD78 Ala4>Glu is constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 23-mer oligonucleotide BB10199(5'-GGTTGGAGTGTCTTCAGCCAAGG-3' SEQ ID NO 86) was used to mutate the LD78gene and a correct clone identified (M13DB126). The mutant gene wascloned into the expression vector to create pDB147. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 42

Design and Construction of LD78 Variant Arg 17>Glu Gln18>Glu (Mutant110) and Construction of an LD78 Arg17>Glu Gln18>Glu Expression Vector

LD78 Arg17>Glu Gln18>Glu is constructed and cloned into the pSW6 yeastexpression vector as described in Example 1. A 22-mer oligonucleotideBB10200 (5'-GGAATTTCTTCAGAGGTGTAAG-3' SEQ ID NO 87) was used to mutatethe LD78 gene and a correct clone identified (M13DB124). The mutant genewas cloned into the expression vector to create pDB140. Expression ofthe mutant LD78 protein was achieved according to methods described inPreparation 3.

Example 43

Design and Construction of LD78 Variant Leu67>Glu (Mutant 111) andConstruction of an LD78 Leu67>Glu Expression Vector

LD78 Leu67>Glu is constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 28-mer oligonucleotide BB10201(5'-CCTTATTAGGCAGATTCTTCCAAGTCAG-3' SEQ ID NO 88) was used to mutate theLD78 gene and a correct clone identified (M13DB125). The mutant gene wascloned into the expression vector to create pDB141. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 44

Design and Construction of LD78 Variant Ser46>Ala (Mutant 95) andConstruction of an LD78 Ser46>Ala Expression Vector

LD78 Ser46>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 20-mer oligonucleotide BB9537(5'-GACTrGTCTAGCGCGCTTAG-3' SEQ ID NO 89) was used to mutate the LD78gene and a correct clone identified (M13DB107). The mutant gene wascloned into the expression vector to create pDB117. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 45

Design and Construction of LD78 Variant Leu2>Ala (Mutant 55) andConstruction of an LD78 Leu2>Ala Expression Vector

LD78 Leu2>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide BB9497(5'-GTCAGCAGCAGCGGATCTT-3' SEQ ID NO 90) was used to mutate the LD78gene and a correct clone identified (pGHC654). The mutant gene wascloned into the expression vector to create pDB102. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 46

Design and Construction of LD78 Variant Ala3>Ser (Mutant 56) andConstruction of an LD78 Ala3>Ser Expression Vector

LD78 Ala3>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 17-mer oligonucleotide BB9498(5'-GTCAGCAGACAAGGATC-3' SEQ ID NO 91) was used to mutate the LD78 geneand a correct clone identified (pGHC655). The mutant gene was clonedinto the expression vector to create pDB123. Expression of the mutantLD78 protein was achieved according to methods described in Preparation3.

Example 47

Design and Construction of LD78 Variant Ala4>Ser (Mutant 57) andConstruction of an LD78 Ala4>Ser Expression Vector

LD78 Ala4>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. An 18-mer oligonucleotide BB9499(5'-GAGTGTCAGAAGCCAAGG-3' SEQ ID NO 92) was used to mutate the LD78 geneand a correct clone identified (pGHC656). The mutant gene was clonedinto the expression vector to create pDB124. Expression of the mutantLD78 protein was achieved according to methods described in Preparation3.

Example 48

Design and Construction of LD78 Variant Leu67>Ala (Mutant 75) andConstruction of an LD78 Leu67>Ala Expression Vector

LD78 Leu67>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 22-mer oligonucleotide BB9517(5'-ATTAGGCAGAGGCTTCCAAGTC-3' SEQ ID NO 93) was used to mutate the LD78gene and a correct clone identified (pRC58/75). The mutant gene wascloned into the expression vector to create pRC59/75. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 49

Design and Construction of LD78 Variant with the First Six Amino AcidsDeleted (N1-6) and Pro7>Ser (Mutant 103) and Construction of an N1-6Pro7>Ser Expression Vector

N1-6 Pro7>Ser LD78 was constructed and cloned into the pSW6 yeastexpression vector as described in Example 1. A 34-mer oligonucleotideBB9781 (5'-GAGAAACAACAAGCGGTAGATCTTTTATCCAAGC-3' SEQ ID NO 94) was usedto mutate the LD78 gene and a correct clone identified (pRC58/103). Themutant gene was cloned in to the expression vector to create pRC59/103.Expression of the mutant LD78 protein was achieved according to methodsdescribed in Preparation 3.

Example 50

Design and Construction of LD78 Variant Phe28>Tyr (Mutant 12) andConstruction of an LD78 Phe28>Tyr Expression Vector

LD78 Phe28>Tyr was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 25-mer oligonucleotide BB6381(5'-GAAGAAGTTTCA(G/C/T)AGTAGTCAGCAA-3' SEQ ID NO 51) was used to mutatethe LD78 gene and a correct clone identified (pGHC611). The mutant genewas cloned into the expression vector to create pRC59/12. Expression ofthe mutant LD78 protein was achieved according to methods described inPreparation 3.

Example 51

Design and Construction of LD78 Variant Asp5>Ser (Mutant 37) andConstruction of an LD78 Asp5>Ser Expression Vector

LD78 Asp5>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 20-mer oligonucleotide BB9430(5'-GTTGGAGTGGAAGCAGCCAA-3' SEQ ID NO 95) was used to mutate the LD78gene and a correct clone identified (pGHC636). The mutant gene wascloned into the expression vector to create pDB134. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 52

Design and Construction of LD78 Variant Phe23>Ala (Mutant 83) andConstruction of an LD78 Phe23>Ala Expression Vector

LD78 Phe23>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. An 18-mer oligonucleotide BB9525(5'-CAGCAATGGCATTTTGTG-3' SEQ ID NO 96) was used to mutate the LD78 geneand a correct clone identified (M13DB119). The mutant gene was clonedinto the expression vector to create pDB137. Expression of the mutantLD78 protein was achieved according to methods described in Preparation3.

Example 53

Design and Construction of LD78 Variant Lys44>Ser (Mutant 42) andConstruction of an LD78 Lys44>Ser Expression Vector

LD78 Lys44>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 21-mer oligonucleotide BB9435(5'-GTCTCGAGCGAGAAGTCAAGA-3' SEQ ID NO 97) was used to mutate the LD78gene and a correct clone identified (pGHC641). The mutant gene wascloned into the expression vector to create pSJE91. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 54

Design and Construction of LD78 Variant Arg45>Ser (Mutant 43) andConstruction of an LD78 Arg45>Ser Expression Vector

LD78 Arg45>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. An 18-mer oligonucleotide BB9436(5'-GTCTCGAGGACTTAGTCA-3' SEQ ID NO 98) was used to mutate the LD78 geneand a correct clone identified (pGHC642). The mutant gene was clonedinto the expression vector to create pRC59/43. Expression of the mutantLD78 protein was achieved according to methods described in Preparation3.

Example 55

Design and Construction of LD78 Variant Glu55>Ser (Mutant 46) andConstruction of an LD78 Glu55>Ser Expression Vector

LD78 Glu55>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 22-mer oligonucleotide BB9423(5'-GAACCCATTCAGAAGATGGGTC-3' SEQ ID NO 99) was used to mutate the LD78gene and a correct clone identified (pGHC645). The mutant gene wascloned into the expression vector to create pSJE95. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 56

Design and Construction of LD78 Variant Glu56>Ser (Mutant 47) andConstruction of an LD78 Glu56>Ser Expression Vector

LD78 Glu56>Ser is constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 21-mer oligonucleotide BB9424(5'-TTTGAACCCAAGATTCAGATG-3' SEQ ID NO 100) is used to mutate the LD78gene. The mutant gene was cloned into the expression vector according tothe method of Example 1 and expression of the mutant LD78 protein wasachieved according to methods described in Preparation 3.

Example 57

Design and Construction of LD78 Variant Lys60>Ser (Mutant 48) andConstruction of an LD78 Lys60>Ser Expression Vector

LD78 Lys60>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 21-mer oligonucleotide BB9425(5'-CAGAAACATAAGATTGAACCC-3' SEQ ID NO 101) was used to mutate the LD78gene and a correct clone identified (pGHC647). The mutant gene wascloned into the expression vector to create pSJE97. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 58

Design and Construction of LD78 Variant Asp64>Ser (Mutant 50) andConstruction of an LD78 Asp64>Ser Expression Vector

LD78 Asp64>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 20-mer oligonucleotide BB9427(5'-CAATTCCAAGGAAGAAACAT-3' SEQ ID NO 102) was used to mutate the LD78gene and a correct clone identified (pGHC649). The mutant gene wascloned into the expression vector to create pSJE99. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 59

Design and Construction of an LD78 Variant With the Five C-terminalAmino Acids Deleted (C65-69) (Mutant 61) and Construction of an C65-69LD78 Expression Vector

C65-59 LD78 was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 17-mer oligonucleotide BB9503(5'-CCTTATTAGTCAGAAAC-3' SEQ ID NO 103) was used to mutate the LD78 geneand a correct clone identified (M13DB113). The mutant gene was clonedinto the expression vector to create pDB103. Expression of the mutantLD78 protein was achieved according to methods described in Preparation3.

Example 60

Design and Construction of LD78 Variant Asp26>Ala Glu29>Arg (Mutant 101)and Construction of an LD78 Asp26>Ala Glu29>Arg Expression Vector

LD78 Asp26>Ala Glu29>Arg was constructed and cloned into the pSW6 yeastexpression vector as described in Example 1. A 33-mer oligonucleotideBB9443 (5'-TTGAGAAGAAGTTCTAAAGTAGGCAGCAATGAA-3' SEQ ID NO 104) was usedto mutate the LD78 gene. The mutant gene was cloned into the expressionvector to create pDB133. Expression of the mutant LD78 protein wasachieved according to methods described in Preparation 3.

Example 61

Design and Construction of LD78 Variant Asp26>Ala Glu29>Arg Arg47>Glu(Mutant 102) and Construction of an LD78 Asp26>Ala Glu29>Arg Arg47>GluExpression Vector

LD78 Asp26>Ala Glu29>Arg Arg47>Glu is constructed and cloned into thepSW6 yeast expression vector essentially as described in Example 1. A33-mer oligonucleotide BB9443 (5'-TTGAGAAGAAGTTCTAAAGTAGGCAGCAATGAA-3'SEQ ID NO 104) was used to mutate the LD78 Arg47>Glu gene (pGHC614 ofExample 11) and a correct clone identified (pRC58/102). The mutant genewas cloned into the expression vector to create pRC59/102. Expression ofthe mutant LD78 protein was achieved according to methods described inPreparation 3.

Example 62

Design and Construction of LD78 Variant Lys36>Ser (Mutant 41) andConstruction of an LD78 Lys36>Ser Expression Vector

LD78 Lys36>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 20-mer oligonucleotide BB9434(5'-GACACCTGGAGAGGAACATT-3' SEQ ID NO 105) was used to mutate the LD78gene and a correct clone identified (pGHC640). The mutant gene wascloned into the expression vector to create pRC59/41. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 63

Design and Construction of LD78 Variant Leu65>Ala (Mutant 51) andConstruction of an LD78 Leu65>Ala Expression Vector

LD78 Leu65>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 22-mer oligonucleotide BB9428(5'-CAGACAATTCAGCGTCAGAAAC-3' SEQ ID NO 106) was used to mutate the LD78gene and a correct clone identified (pGHC650). The mutant gene wascloned into the expression vector to create pSJE100. Expression of themutant LD79 protein was achieved according to methods described inPreparation 3.

Example 64

Design and Construction of LD78 Variant Glu66>Ser (Mutant 52) andConstruction of an LD78 Glu66>Ser Expression Vector

LD78 Glu66>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 20-mer oligonucleotide BB9429(5'-GGCAGACAAAGACAAGTCAG-3' SEQ ID NO 107) was used to mutate the LD78gene and a correct clone identified (pGHC651). The mutant gene wascloned into the expression vector to create pSJE101. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 65

Design and Construction of LD78 Variant Ala69>Ser (Mutant 53) andConstruction of an LD78 Ala69>Ser Expression Vector

LD78 Ala69>Ser is constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide BB9495(5'-CTFATTAGGAAGACAATTC-3' SEQ ID NO 108) was used to mutate the LD78gene and a correct clone identified (pRC58/53). The mutant gene wascloned into the expression vector to create pRC59/53. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 66

Design and Construction of LD78 Variant Ser1>Ala (Mutant 54) andConstruction of an LD78 Ser1>Ala Exxpression Vector

LD78 Ser1>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide BB9496(5'-CAGCCAAGGCTCTTTTATC-3' SEQ ID NO 109) was used to mutate the LD78gene and a correct clone identified (pGHC653). The mutant gene wascloned into the expression vector to create pDB101. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 67

Design and Construction of LD78 Variant Gln33>Ser (Mutant 67) andConstruction of an LD78 Gln33>Ser Expression Vector

LD78 Gln33>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 20-mer oligonucleotide BB9509(5'-CTTGGAACAAGAAGAAGAAG-3' SEQ ID NO 110) was used to mutate the LD78gene and a correct clone identified (M13DB127). The mutant gene wascloned into the expression vector to create pDB143. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 68

Design and Construction of LD78 Variant Tyr61>Ala (Mutant 73) andConstruction of an LD78 Tyr61>Ala Expression Vector

LD78 Tyr61>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide BB9515(5'-GTCAGAAACAGCTTTTTGA-3' SEQ ID NO 111) was used to mutate the LD78gene and a correct clone identified (M13DB115). The mutant gene wascloned into the expression vector to create pDB106. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 69

Design and Construction of LD78 Variant Ser31>Ala (Mutant 87) andConstruction of an LD78 Ser31>Ala Expression Vector

LD78 Ser31>Ala was constructed and is cloned into the pSW6 yeastexpression vector as described in Example 1. A 19-mer oligonucleotideBB9529 (5'-CATTGAGAAGCAGTTTCAA-3' SEQ ID NO 112) was used to mutate theLD78 gene and a correct clone identified (pGHC686). The mutant gene wascloned into the expression vector to create pDB132. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 70

Design and Construction of LD78 Variant Ser32>Ala (Mutant 88) andConstruction of an LD78 Ser32>Ala Expression Vector

LD78 Ser32>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide BB9530(5'-GAACATTGAGCAGAAGTTT-3' SEQ ID NO 113) was used to mutate the LD78gene and a correct clone identified (M13DB101). The mutant gene wascloned into the expression vector to create pDB110. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 71

Design and Construction of LD78 Vanrant Leu42>Ala (Mutant 94) andConstruction of an LD78 Leu42>Ala Expression Vector

LD78 Leu42>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 21-mer oligonucleotide BB9536(5'-GCGCTTAGTAGCGAAGATGAC-3' SEQ ID NO 114) was used to mutate the LD78gene and a correct clone identified (M13DB106). The mutant gene wascloned into the expression vector to create pDB116. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 72

Design and Construction of LD78 Variant Asp52>Ser (Mutant 45) andConstruction of an LD78 Asp52>Ser Expression Vector

LD78 Asp52>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 22-mer oligonucleotide BB9422(5'-CTTCAGATGGAGAAGCACAGAC-3' SEQ ID NO 115) was used to mutate the LD78gene and a correct clone identified (pGHC644). The mutant gene wascloned into the expression vector to create pSJE94. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 73

Design and Construction of LD78 Variant Val62>Ala (Mutant 49) andConstruction of an LD78 Val62>Ala Expression Vector

LD78 Val62>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 21-mer oligonucleotide BB9426(5'-CAAGTCAGAAGCATATTTTTG-3' SEQ ID NO 116) was used to mutate the LD78gene and a correct clone identified (pGHC648). The mutant gene wascloned into the expression vector to create pDB100. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 74

Design and Construction of LD73 Variant Ser13>Ala (Mutant 62) andConstruction of an LD78 Ser13>Ala Expression Vector

LD78 Ser13>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 17-mer oligonucleotide BB9504(5'-GGTGTAAGCGAAACAAC-3' SEQ ID NO 117) was used to mutate the LD78 geneand a correct clone identified (pGHC661). The mutant gene was clonedinto the expression vector to create pSJE111. Expression of the mutantLD78 protein was achieved according to methods described in Preparation3.

Example 75

Design and Construction of LD78 Variant Ser16>Ala (Mutant 63) andConstruction of an LD78 Ser16>Ala Expression Vector

LD78 Ser16>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide BB9505(5'-ATTTGTCTAGCGGTGTAAG-3' SEQ ID NO 118) was used to mutate the LD78gene and a correct clone identified (pGHC662). The mutant gene wascloned into the expression vector to create pSJE112. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 76

Design and Construction of LD78 Variant Pro20>Ala (Mutant 65) andConstruction of an LD78 Pro20>Ala Expression Vector

LD78 Pro20>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 20-mer oligonucleotide BB9507(5'-GAAATTTTGAGCAATTTGTC-3' SEQ ID NO 119) was used to mutate the LD78gene and a correct clone identified (pGHC664). The mutant gene wascloned into the expression vector to create pDB104. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 77

Design and Construction of LD78 Variant Ser35>Ala (Mutant 68) andConstruction of an LD78 Ser35>Ala Expression Vector

LD78 Ser35>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. An 18-mer oligonucleotide BB9510(5'-CTGGCTTGGCACATTGAG-3' SEQ ID NO 120) was used to mutate the LD78gene and a correct clone identified (pGHC668). The mutant gene wascloned into the expression vector to create pSJE117. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 78

Design and Construction of LD78 Variant Gln59>Ser (Mutant 72) andConstruction of an LD78 Gln59>Ser Expression Vector

LD78 Gln59>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 23-mer oligonucleotide BB9514(5'-GAAACATATTTAGAAACCCATTC-3' SEQ ID NO 121) was used to mutate theLD78 gene and a correct clone identified (M13DB114). The mutant gene wascloned into the expression vector to create pDB105. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 79

Design and Construction of LD78 Variant Ser68>Ala (Mutant 76) andConstruction of an LD78 Ser68>Ala Expression Vector

LD78 Ser68>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide BB9518(5'-ATTAGGCAGCCAATTCCAA-3' SEQ ID NO 122) was used to mutate the LD78gene and a correct clone identified (M13DB100). The mutant gene wascloned into the expression vector to create pDB107. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 80

Design and Construction of LD78 Variant Tyr14>Ala (Mutant 78) andConstruction of an LD78 Tyr14>Ala Expression Vector

LD78 Tyr14>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide BB9520(5'-CTAGAGGTGGCAGAGAAAC-3' SEQ ID NO 123) was used to mutate the LD78gene and a correct clone identified (M13DB116). The mutant gene wascloned into the expression vector to create pDB108. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 81

Design and Construction of LD78 Variant Ile19>Ala (Mutant 80) andConstruction of an LD78 Ile19>Ala Expression Vector

LD78 Ile19>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide BB9522(5'-TTTTGTGGAGCTTGTCTAG-3' SEQ ID NO 124) was used to mutate the LD78gene and a correct clone identified (M13DB117). The mutant gene wascloned into the expression vector to create pDB109. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 82

Design and Construction of LD78 Variant Pro37>Ala (Mutant 89) andConstruction of an LD78 Pro37>Ala Expression Vector

LD78 Pro37>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 20-mer oligonucleotide BB9531(5'-GATGACACCAGCCTTGGAAC-3' SEQ ID NO 125) was used to mutate the LD78gene and a correct clone identified (M13DB102). The mutant gene wascloned into the expression vector to create pDB111. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 83

Design and Construction of LD78 Variant Gly38>Ala (Mutant 90) andConstruction of an LD78 Gly38>Ala Expression Vector

LD78 Gly38>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Exarnple 1. A 20-mer oligonucleotide BB9532(5'-GAAGATGACAGCTGGCTTGG-3' SEQ ID NO 126) was used to mutate the LD78gene and a correct clone identified (M13DB103). The mutant gene wascloned into the expression vector to create pDB112. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 84

Design and Construction of LD78 Variant Val39>Ala (Mutant 91) andConstruction of an LD78 Val39>Ala Expression Vector

LD78 Val39>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. An 18-mer oligonucleotide BB9533(5'-AGAAGATGGCACCTGGCT-3' SEQ ID NO 127) was used to mutate the LD78gene and a correct clone identified (M13DB118). The mutant gene wascloned into the expression vector to create pDB113. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 85

Design and Construction of LD78 Variant Thr6>Ala (Mutant 58) andConstruction of an LD78 Thr6>Ala Expression Vector

LD78 Thr6>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 17-mer oligonucleotide BB9500(5'-GGTTGGAGCGTCAGCAG-3' SEQ ID NO 128) was used to mutate the LD78 geneand a correct clone identified (pRC58/58). The mutant gene was clonedinto the expression vector to create pRC59/58. Expression of the mutantLD78 protein was achieved according to methods described in Preparation3.

Example 86

Design and Construction of LD78 Variant Gln21>Ser (Mutant 81) andConstruction of an LD78 Gln21>Ser Expression Vector

LD78 Gln21>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 22-mer oligonucleotide BB9523(5'-CAATGAAATTAGATGGAATTTG-3' SEQ ID NO 129) was used to mutate the LD78gene and a correct clone identified (M13DB118). The mutant gene wascloned into the expression vector to create pDB136. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 87

Design and Construction of LD78 Variant Thr43>Ala (Mutant 69) andConstruction of an LD78 Thr43>Ala Expression Vector

LD78 Thr43>Ala was constructed and is cloned into the pSW6 yeastexpression vector as described in Example 1. A 17-mer oligonucleotideBB9511 (5'-GCGCTTAGCCAAGAAGA-3' SEQ ID NO 130) was used to mutate theLD78 gene and a correct clone identified (pGHC669). The mutant gene wascloned into the expression vector to create pRC59/69. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 88

Design and Construction of LD78 Variant Pro7>Ala (Mutant 59) andConstruction of an LD78 Pro7>Ala Expression Vector

LD78 Pro7>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide BB9501(5'-CAAGCGGTAGCAGTGTCAG-3' SEQ ID NO 131) was used to mutate the LD78gene and a correct clone identified (pGHC658). The mutant gene wascloned into the expression vector to create pDB125. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 89

Design and Construction of LD78 Variant Thr8>Ala (Mutant 60) andConstruction of an LD78 Thr8>Ala Expression Vector

LD78 Thr8>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. An 18-mer oligonucleotide BB9502(5'-ACAAGCGGCTGGAGTGTC-3' SEQ ID NO 132) was used to mutate the LD78gene and a correct clone identified (pRC58/60). The mutant gene wascloned into the expression vector to create pRC59/60. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 90

Design and Construction of LD78 Variant Tyr27>Ala (Mutant 66) andConstruction of an LD78 Tyr27>Ala Expression Vector

LD78 Tyr27>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. An 18-mer oligonucleotide BB9508(5'-GTTTCAAAGGCGTCAGCA-3' SEQ ID NO 133) was used to mutate the LD78gene and a correct clone identified (pGHC665). The mutant gene wascloned into the expression vector to create pDB126. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 91

Design and Construction of LD78 Variant Pro53>Ala (Mutant 71) andConstruction of an LD78 Pro53>Ala Expression Vector

LD78 Pro53>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 20-mer oligonucleotide BB9513(5'-TTCTTCAGATGCGTCAGCAC-3' SEQ ID NO 134) was used to mutate the LD78gene and a correct clone identified (pRC58/71). The mutant gene wascloned into the expression vector to create pRC59/71. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 92

Design and Construction of LD78 Variant Ser63>Ala (Mutant 74) andConstruction of an LD78 Ser63>Ala Expression Vector

LD78 Ser63>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. An 18-mer oligonucleotide BB9516(5'-CAAGTCAGCAACATATTT-3' SEQ ID NO 135) was used to mutate the LD78gene and a correct clone identified (pGHC674). The mutant gene wascloned into the expression vector to create pDB145. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 93

Design and Construction of LD78 Variant Thr15>Ala (Mutant 79) andConstruction of an LD78 Thr15>Ala Expression Vector

LD78 Thr15>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 17-mer oligonucleotide BB9521(5'-GTCTAGAGGCGTAAGAG-3' SEQ ID NO 136) was used to mutate the LD78 geneand a correct clone identified (pGHC678). The mutant gene was clonedinto the expression vector to create pDB128. Expression of the mutantLD78 protein was achieved according to methods described in Preparation3.

Example 94

Design and Construction of LD78 Variant Asn22>Ser (Mutant 82) andConstruction of an LD78 Asn22>Ser Expression Vector

LD78 Asn22>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. An 18-mer oligonucleotide BB9524(5'-CAATGAAAGATTGTGGAA-3' SEQ ID NO 137) was used to mutate the LD78gene and a correct clone identified (pGHC681). The mutant gene wascloned into the expression vector to create pDB129. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 95

Design and Construction of LD78 Variant Ala25>Ser (Mutant 84) andConstruction of an LD78 Ala25>Ser Expression Vector

LD78 Ala25>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 17-mer oligonucleotide BB9526(5'-GTAGTCAGAAATGAAAT-3' SEQ ID NO 138) was used to mutate the LD78 geneand a correct clone identified (pRC58/84). The mutant gene was clonedinto the expression vector to create pRC59/84. Expression of the mutantLD78 protein was achieved according to methods described in Preparation3.

Example 96

Design and Construction of LD78 Variant Thr30>Ala (Mutant 86) andConstruction of an LD78 Thr30>Ala Expression Vector

LD78 Thr30>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. An 18-mer oligonucleotide BB9528(5'-GAGAAGAAGCTTCAAAGT-3' SEQ ID NO 139) was used to mutate the LD78gene and a correct clone identified (pGHC685). The mutant gene wascloned into the expression vector to create pDB131. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 97

Design and Construction of LD78 Variant Phe41>Ala (Mutant 93) andConstruction of an LD78 Phe41>Ala Expression Vector

LD78 Phe41>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 20-mer oligonucleotide BB9535 (5'CTTAGTCAAGGCGATGACAC-3' SEQ ID NO 140) was used to mutate the LD78 geneand a correct clone identified (M13DB105). The mutant gene was clonedinto the expression vector to create pDB115. Expression of the mutantLD78 protein was achieved according to methods described in Preparation3.

Example 98

Design and Construction of LD78 Variant Val49>Ala (Mutant 96) andConstruction of an LD78 Val49>Ala Expression Vector

LD78 Val49>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 20-mer oligonucleotide BB9538(5'-GTCAGCACAGGCTTGTCTCG-3' SEQ ID NO 141) was used to mutate the LD78gene and a correct clone identified (M13DB108). The mutant gene wascloned into the expression vector to create pDB118. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 99

Design and Construction of LD78 Variant Ala51>Ser (Mutant 97) andConstruction of an LD78 Ala51>Ser Expression Vector

LD78 Ala51>Ser was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 17-mer oligonucleotide BB9539(5'-TGGGTCAGAACAGACTT-3' SEQ ID NO 142) was used to mutate the LD78 geneand a correct clone identified (M13DB109). The mutant gene was clonedinto the expression vector to create pDB119. Expression of the mutantLD78 protein was achieved according to methods described in Preparation3.

Example 100

Design and Construction of LD78 Variant Ser54>Ala (Mutant 98) andConstruction of an LD78 Ser54>Ala Expression Vector

LD78 Ser54>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide BB9540(5'-CATTCTTCAGCTGGGTCAG-3' SEQ ID NO 143) was used to mutate the LD78gene and a correct clone identified (M13DB110). The mutant gene wascloned into the expression vector to create pDB120. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 101

Design and Construction of LD78 Variant Trp57>Ala (Mutant 99) andConstruction of an LD78 Trp57>Ala Expression Vector

LD78 Trp57>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 20-mer oligonucleotide BB9541(5'-ATTTTTGAACAGCTTCTTCA-3' SEQ ID NO 144) was used to mutate the LD78gene and a correct clone identified (M13DB111). The mutant gene wascloned into the expression vector to create pDB121. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 102

Design and Construction of LD78 Variant Val58>Ala (Mutant 100) andConstruction of an LD78 Val58>Ala Expression Vector

LD78 Val58>Ala was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 22-mer oligonucleotide BB9542(5'-CATATTTTTGAGCCCATTCTTC-3' SEQ ID NO 145) was used to mutate the LD78gene and a correct clone identified (M13DB132). The mutant gene wascloned into the expression vector to create pDB122. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 103

Design and Construction of LD78 Variant Trp57>Leu (Mutant 112) andConstruction of an LD78 Trp57>Leu Expression Vector

LD78 Trp57>Leu was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 21-mer oligonucleotide BB10374(5'-TTTTTGAACCAATTCTTCAGA-3' SEQ ID NO 146) was used to mutate the LD78gene and a correct clone identified (pRC58/112). The mutant gene wascloned into the expression vector to create pRC59/112. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 104

Design and Construction of LD78 Variant Lys60>Asp (Mutant 113) andConstruction of an LD78 Lys60>Asp Expression Vector

LD78 Lys60>Asp was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 21-mer oligonucleotide BB10375(5'-CAGAAACATAATCTTGAACCC-3' SEQ ID NO 147) was used to mutate the LD78gene and a correct clone identified (pRC58/113). The mutant gene wascloned into the expression vector to create pDB142. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 105

Design and Construction of LD78 Variant Tyr61>Asp (Mutant 114) andConstruction of an LD78 Tyr61>Asp Expression Vector

LD78 Tyr61>Asp was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide BB10376(5'-GTCAGAAACATCTTTTTGA-3' SEQ ID NO 148) was used to mutate the LD78gene and a correct clone identified (pRC58/114). The mutant gene wascloned into the expression vector to create pRC59/114. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 106

Design and Construction of LD78 Variant Phe12>Asp (Mutant 115) andConstruction of an LD78 Phe12>Asp Expression Vector

LD78Phe12>Asp was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide BB10377(5'-GTGTAAGAATCACAACAAG-3' SEQ ID NO 149) was used to mutate the LD78gene and a correct clone identified (pRC58/115). The mutant gene wascloned into the expression vector to create pRC59/115. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 107

Design and Construction of LD78 Variant Thr8>Glu (Mutant 116) andConstruction of an LD78 Thr8>Glu Expression Vector

LD78 Thr8>Glu is constructed and is cloned into the pSW6 yeastexpression vector as described in Example 1. A 23-mer oligonucleotideBB11235 (5'-GAAACAACAAGCTTCTGGAGTGT-3' SEQ ID NO 150) is used to mutatethe LD78 gene. The mutant gene is cloned into the expression vectoraccording to the methods of Example 1 and expression of the mutant LD78protein is achieved according to methods described in Preparation 3.

Example 108

Design and Construction of LD78 Variant Ser68>Glu (Mutant 117) andConstruction of an LD78 Ser68>Glu Expression Vector

LD78 Ser68>Glu was constructed and is cloned into the pSW6 yeastexpression vector as described in Example 1. A 19-mer oligonucleotideBB10379 (5'-ATTAGGCTTCCAATTCCAA-3' SEQ ID NO 151) was used to mutate theLD78 gene and a correct clone identified (pRC58/117). The mutant gene iscloned into the expression vector according to the methods of Example 1and expression of the mutant LD78 protein is achieved according tomethods described in Preparation 3.

Example 109

Design and Construction of LD78 Variant Leu67>Asp (Mutant 118) andConstruction of an LD78 Leu67>Asp Expression Vector

LD78 Leu67>Asp was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 22-mer oligonucleotide BB10380(5'-ATTAGGCAGAATCTTCCAAGTC-3' SEQ ID NO 152) was used to mutate the LD78gene and a correct clone identified (M13DB130). The mutant gene wascloned into the expression vector to create pDB148. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 110

Design and Construction of LD78 Variant Asp64>Arg (Mutant 119) andConstruction of an LD78 Asp64>Arg Expression Vector

LD78 Asp64>Arg was constructed and was cloned into the pSW6 yeastexpression vector as described in Example 1. A 20-mer oligonucleotideBB10381 (5'-CAATTCCAATCTAGAAACAT-3' SEQ ID NO 153) is used to mutate theLD78 gene and a correct clone identified (pGHC569). The mutant gene iscloned into the expression vector according to the methods of Example 1and expression of the mutant LD78 protein is achieved according tomethods described in Preparation 3.

Example 111

Design and Construction of LD78 Variant Ser31>Glu (Mutant 120) andConstruction of an LD78 Ser31>Glu Expression Vector

LD78 Ser31>Glu is constructed and is cloned into the pSW6 yeastexpression vector as described in Example 1. A 19-mer oligonucleotideBB10382 (5'-CATTGAGATTCAGTTTCAA-3' SEQ ID NO 154) is used to mutate theLD78 gene. The mutant gene is cloned into the expression vectoraccording to the methods of Example 1 and expression of the mutant LD78protein is achieved according to methods described in Preparation 3.

Example 112

Design and Construction of LD78 Variant Ile40>Asn (Mutant 121) andConstruction of an LD78 Ile40>Asn Expression Vector

LD78 Ile40>Asn was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide BB10383(5'-GTCAAGAAGTTGACACCTG-3' SEQ ID NO 155) was used to mutate the LD78gene and a correct clone identified (pRC58/121). The mutant gene wascloned into the expression vector to create pRC59/121. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 113

Design and Construction of LD78 Variant Leu42>Asn (Mutant 122) andConstruction of an LD78 Leu42>Asn Expression Vector

LD78 Leu42>Asn was constructed and was cloned into the pSW6 yeastexpression vector as described in Example 1. A 21-mer oligonucleotideBB10964 (5'-GCGCTTAGTGTTGAAGATGAC-3' SEQ ID NO 156) is used to mutatethe LD78 gene and a correct clone identified (pGHC568). The mutant geneis cloned into the expression vector according to the methods of Example1 and expression of the mutant LD78 protein is achieved according tomethods described in Preparation 3.

Example 114

Design and Construction of LD78 Variant Cys10, Cys11>Cys-Gln-Cys (Mutant123) and Construction of an LD78 Cys10, Cys11>Cys-Gln-Cys ExpressionVector

LD78 Cys10, Cys11>Cys-Gln-Cys was constructed and cloned into the pSW6yeast expression vector as described in Example 1. A 27-meroligonucleotide BB10385 (5'-GTAAGAGAAACATTGACAAGCGGTTGG-3' SEQ ID NO157) was used to mutate the LD78 gene and a correct clone identified(pRC58/123). The mutant gene was cloned into the expression vector tocreate pRC59/123. Expression of the mutant LD78 protein was achievedaccording to methods described in Preparation 3.

Example 115

Design and Construction of LD78 Variant Glu55>Gln, Glu56>Gln (Mutant124) and Construction of an LD78 Glu55>Gln, Glu56>Gln Expression Vector

LD78 Glu55>Gln, Glu56>Gln is constructed and is cloned into the pSW6yeast expression vector as described in Example 1. A 24-meroligonucleotide BB10386 (5'-TTGAACCCATTGTTGAGATGGGTC-3' SEQ ID NO 158)is used to mutate the LD78 gene. The mutant gene is cloned into theexpression vector according to the methods of Example 1 and expressionof the mutant LD78 protein is achieved according to methods described inPreparation 3.

Example 116

Design and Construction of LD78 Variant Asp26>Gln (Mutant 125) andConstruction of an LD78 Asp26>Gln Expression Vector

LD78 Asp26>Gln was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 21-mer oligonucleotide BB10529(5'-GTTTCAAAGTATTGAGCAATG-3' SEQ ID NO 159) was used to mutate the LD78gene and a correct clone identified (pRC58/125). The mutant gene wascloned into the expression vector to create pRC59/125. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 117

Design and Construction of LD78 Variant Lys36>Glu (Mutant 126) andConstruction of an LD78 Lys36>Glu Expression Vector

LD78 Lys36>Glu was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 26-mer oligonucleotide BB10530(5'-GATGACACCTGGTTCGGAACATTGAG-3' SEQ ID NO 160) was used to mutate theLD78 gene and a correct clone identified (pRC58/126). The mutant genewas cloned into the expression vector to create pRC59/126. Expression ofthe mutant LD78 protein was achieved according to methods described inPreparation 3.

Example 118

Design and Construction of LD78 Variant Lys44>Glu (Mutant 127) andConstruction of an LD78 Lys44>Glu Expression Vector

LD78 Lys44>Glu was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 26-mer oligonucleotide BB10531(5'-CTTGTCTCGAGCGTTCAGTCAAGAAG-3' SEQ ID NO 161) was used to mutate theLD78 gene and a correct clone identified (pRC58/127). The mutant genewas cloned into the expression vector to create pRC59/127. Expression ofthe mutant LD78 protein was achieved according to methods described inPreparation 3.

Example 119

Design and Construction of LD78 Variant Arg45>Glu (Mutant 128) andConstruction of an LD78 Arg45>Glu Expression Vector

LD78 Arg45>Glu was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 25-mer oligonucleotide BB10532(5'-GACTTGTCTCGATTCCTTAGTCAAG-3' SEQ ID NO 162) was used to mutate theLD78 gene and a correct clone identified (pRC58/128). The mutant genewas cloned into the expression vector to create pRC59/128. Expression ofthe mutant LD78 protein was achieved according to methods described inPreparation 3.

Example 120

Design and Construction of LD78 Variant Asp52>Gln (Mutant 129) andConstruction of an LD78 Asp52>Gln Expression Vector

LD78 Asp52>Gln was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 27-mer oligonucleotide BB10533(5'-CCATTCTTCAGATGGTGGAGCACAGAC-3' SEQ ID NO 163) was used to mutate theLD78 gene and a correct clone identified (M13DB131). The mutant gene wascloned into the expression vector to create pDB149. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 121

Design and Construction of LD78 Variant Glu66>Gln (Mutant 130) andConstruction of an LD78 Glu66>Gln Expression Vector

LD78 Glu66>Gln was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 19-mer oligonucleotide BB10534(5'-GCAGACAATTGCAAGTCAG-3' SEQ ID NO 164) was used to mutate the LD78gene and a correct clone identified (pRC58/130). The mutant gene wascloned into the expression vector to create pRC59/130. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 122

Design and Construction of LD78 Variant Ile24>Leu (Mutant 131) andConstruction of an LD78 Ile24>Leu Expression Vector

LD78 Ile24>Leu was constructed and is cloned into the pSW6 yeastexpression vector as described in Example 1. A 21-mer oligonucleotideBB10535 (5'-GTAGTCAGCCAAGAAATTTTG-3' SEQ ID NO 165) was used to mutatethe LD78 gene and a correct clone identified (M13DB128). The mutant geneis cloned into the expression vector according to the methods of Example1 and expression of the mutant LD78 protein is achieved according tomethods described in Preparation 3.

Example 123

Design and Construction of LD78 Variant Ile24>Val (Mutant 132) andConstruction of an LD78 Ile24>Val Expression Vector

LD78 Ile24>Val was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 21-mer oligonucleotide BB10536(5'-GTAGTCAGCGACGAAATTTTG-3' SEQ ID NO 166) was used to mutate the LD78gene and a correct clone identified (M13DB129). The mutant gene wascloned into the expression vector to create pDB150. Expression of themutant LD78 protein was achieved according to methods described inPreparation 3.

Example 124

Design and Construction of LD78 Variant Arg17>Glu (Mutant 133) andConstruction of an LD78 Arg17>Glu Expression Vector

LD78 Arg17>Glu was constructed and cloned into the pSW6 yeast expressionvector as described in Example 1. A 22-mer oligonucleotide BB10195(5'-GGAATTMGTTCAGAGGTGTAAG-3' SEQ ID NO 167) was used to mutate the LD78gene and a correct clone identified (pGHC567). The mutant gene is clonedinto the expression vector according to the methods of Example 1 andexpression of the mutant LD78 protein is achieved according to methodsdescribed in Preparation 3.

Example 125

Primary screening of LD78 mutants to identify non-multimerising variantmolecules

In order to screen mutant LD78 molecules for non-multimerisingproperties, supernatants of the expressed constructs were initiallyanalysed by native PAGE and the molecular weight of the LD78 variantprotein identified by immunoblot with a rabbit anti-MIP-1α polyclonalantiserum.

Mutant constructs (described in Examples 1-124) were expressed and grownin shake-flasks according to the methods described in Preparation 3. 100μl aliquots of the culture supernatant were dried using a SPEEDIVAC™concentrator and reconstituted in 7 μl of native PAGE sample buffer (25mM Tris, 10% glycerol, 0.02% bromophenol blue). 5 μl of sample wasloaded onto a 5-50% GRADIPORE HYLINX™ native acrylamide gel (Flowgen)together with high molecular weight range RAINBOW™ markers (AmershamInternational plc, Amersham Place, Little Chalfont, Amersham, Bucks HP79NA) and standard LD78 (purified as described in Preparation 4). The gelwas electrophoresed at 100 volts for 15 minutes according to themanufacturers' instructions as detailed in Comparative Example 3. Thegel was then sandwiched between sheets of nitrocellulose andelectroblotted at 100 mV for 30 minutes in 125 mM Tris, 20 mM glycine,10% methanol, pH8.8 buffer.

After the protein was blotted onto the nitrocellulose membrane, themembrane was placed in 20 ml of blocking buffer (0.5% casein, 154 mMNaCl, 20 mM Tris pH7.4, 0.05% Triton) for 30 minutes at roomtemperature. The membrane was then incubated for 30 minutes at roomtemperature with a 1:2,000 (v/v) dilution (in blocking buffer) of theprimary antibody (polyclonal rabbit anti-MIP-1α produced using standardtechniques) with gentle rotation. The membrane was then given 3×5 minutewashes in blocking buffer. Following the last wash, the membrane wasincubated with a 1:10,000 (v/v) dilution (in blocking buffer) of thesecondary antibody goat anti-rabbit peroxidase (Sigma) for 30 minuteswith gentle rotation at room temperature. Following this incubation, themembrane was given 3 successive 5 minute washes with 150 mM phosphatebuffered saline, pH7.4, and developed as described in ComparativeExample 2.

Whilst this system proved a useful screen, estimates of molecular weightcould not be made due to the sharp (5-50%) acrylamide gradient requiredto effect separation of a broad mass range.

In order to focus more directly on the mass range expected fornon-multimerising variants (8,000-100,000 daltons), 12% acrylamideBIORAD™ native gels (pre-cast in 0.375M Tris-HCl pH8.8, electrophoresisas per manufacturers' instructions with 25 mM Tris, 192 mM glycine,pH8.3, running buffer) have been used to screen both expressedsupernatants and Q-SEPHAROSE™ purified (method described in Preparation4) LD78 variants prepared for electrophoresis as described above. Thegels were electrophoresed at 150 V for 60 minutes and thenelectroblotted or stained as described above. A coomassie stainedexample of the results obtained in this native PAGE system as shown inFIGS. 12 and 13. The results clearly demonstrate (from the mobility ofprotein bands during electrophoresis) that LD78(Glu44;Gln45, (mutant 2of Example 2)), LD78(Glu47, (mutant 15 of Example 11)) andLD78(Glu28;Glu47, (mutant 26 of Example 16)), LD78(Ser17;Glu18 (mutant30 of Example 20)) and LD78(Gln12 (mutant 11 of Example 8)) do not formlarge multimeric complexes. The results in FIGS. 12 and 13 also showthat LD78(Ala26 (mutant 10 of Example 7)), LD78(Glu48 (mutant 1 ofExample 1)), LD78(Glu18 (mutant 29 of Example 19)) and LD78(Ser66(mutant 52 of Example 64)) have increased mobility of electrophoresis,suggesting non-wild-type multimerisation. Known LD78 variants such asLD78 (Leu, Ser, Ala, Prol, Ser38, Gly46, (mutant 35 of Example 25)) andLD78(Ala, Pro1 (mutant 34 of Example 24)) were observed to have the samelow mobility, high molecular weight bands as the wild type control.

Table 1 details the results of the native PAGE primary analysis of LD78variants where those identified to have non-wild type multimerisationproperties are classed as "small", "mix" or "large" according to theclassification in Table 1. Some variants were expressed very poorly andcould not be definitively examined. Variants in Examples 1 to 124 notlisted in Table 1 showed wild type electrophoretic mobility. It isprobable in these cases that the mutated sites are key structuralresidues leading to destabilization of the protein. Selected variantsidentified in this screen were purified to >95% homogeneity (asdescribed in preparation 4) for analysis by SEC and analyticalultracentrifugation as described in Comparative Example 3. The resultsof these analyses are detailed in subsequent examples.

It should be noted that the gel screen does not always tally with theanalytical centrifuge data (see for example LD78 (Glu 48) mutant 1 inExample 135), and that selection of optimum embodiments of the inventionshould preferably not be undertaken on the basis of gel screen dataalone. Possible scientific rationales for this are: (i) in the gelscreen TRIS glycine buffers may chelate metal ions, thus destabilisingaggregation and (ii) changing the number and/or type of charged sidechains can affect the mass/charge ratio on electrophoresis. Also, theconcentration used in the analytical ultracentrifuge assays is 0.5mg/ml, which is not the case in the gel screen assays shown.

                                      TABLE 1    __________________________________________________________________________                Size by                Native    # Mutation  PAGE  SEC kDa                           M.sub.w.sup.o kDa                                 M.sub.w ζ = 0                                      M.sub.w ζ = 1    __________________________________________________________________________    Wt LD78     Large Wt                      Excl 160   10   >250    1 Glu-48 (E135)                Large 131(excl)                           400   100  600    2 Glu-44; Glu-45 (E128)                Small 21   16-5  --   --    5 Ser-17 (E129)                Small 29   57.5  30   100*    10 Ala-26 (E133)                Large 24.5 30    --   --*    11 Glu-12 (E132)                Small 12.8 98    45   140    15 Glu-47 (E127)                Small 21   17.5  --   --    17 Glu-28 (E153)                Small n.d. n.d.  n.d. n.d.    24 Asn-24 (E153)                Small n.d. n.d.  n.d. n.d.    25 Glu-28, Glu-48 (E136)                Small 60   n.d.  n.d. n.d.    26 Glu-28, Glu-47 (E126)                Small 21   15    --   --    28 Arg-29 (E134)                Small 24   n.d.  n.d. n.d.    29 Glu-18 (E130)                Large 48.5 130   75   170    30 Ser-17; Glu-18 (E131)                Mix*  25   41    37   50    34 (E24) (154)                Small Excl 350   260  480    35 (E25) (154)                Small Excl 155   90   200    42 Ser-44 (E138)                Small --   45    35   48    43 Ser-45 (E137)                Small --   25    --   --    51 Ala-65 (E139)                Large --   120   --   --    52 Ser-66 (E140)                Large --   27    --   --    59 Ala-7 (E141)                Small --   1000  400  1200    64 Ser-18 (E142)                Large --   200   --   --    73 Ala-61 (E143)                Mix*  --   400   --   --    77 Ala-12 (E147)                Small --   150   110  170    79 Ala-15 (E150)                Small --   180   110  250    80 Ala-19 (E144)                Mix*  --   6     --   --    81 Ser-21 (E152)                Large --   112   --   --    91 Ala-39 (E145)                Mix*  --   8.2   --   --    109 Glu-5 (E149)                Small --   72    --   --    115 Asp-12 (E148)                Small --   30    --   --    126 Glu-36 (E151)                Small --   200   100  250    MIP-1α                Large Wt                      Excl 310   230  350    110 Glu17;Glu18 (E146)                Small --   30    --   --    __________________________________________________________________________     M.sub.w.sup.o = Whole cell weight average molecular weight     M.sub.w ζ = 0 Point average molecular weight at the meniscus     M.sub.w ζ = 1 Point average molecular weight at the cell base  where     no values for M.sub.w ζ = 0 and M.sub.w ζ = 1 are given with     M.sub.w.sup.o, the sample is monodisperse     E = Example No. of size analysis     * = large and small species present     Large Wt = Electrophoretic mobility on Native PAGE equivalent to Wt LD78     Large = Minor increase in electrophoretic mobility compared to Wt LD78 on     Native PAGE     Small = Major increase in electrophoretic mobility compared to Wt LD78 on     Native PAGE     Extl = Excluded from the SEC gel matrix

Example 126

Mutation of residues Phe28 to Glu and Arg47 to Glu prevents theassociation of LD78 dimers to form tetramers

As detailed in Table 1, pure LD78(Glu28;Glu47, (mutant 26 of Example16)) protein has been studied in 150 mM PBS pH7.4 buffer using SizeExclusion Chromatography on SUPERDEX 75™ resin and by SedimentationEquilibrium with wild type LD78 for comparison. The SEC profile (FIG.14) of this LD78 mutant is a single, symmetrical peak demonstrating adefined, homogenous population of 21 kDa mass. Analysis of thesedimentation equilibrium data shows that LD78(Glu28;Glu47) exists as amonodisperse population of protein species with a mass (M.sup.∘_(w)) of15.2 kDa corresponding to dimers.

The 21 kDa mass measured by SEC in 150 mM PBS pH7.4 is close to thatexpected for an LD78 trimer. The model of association for this moleculedoes not predict formation of this mass species. The absolute massdetermination by sedimentation equilibrium in this buffer gives veryprecise data proving the molecule is a single molecular species of 15.2kDa. The anomalously high mass by SEC arises due to the asymmetricalshape of the LD78 dimer causing non-ideal (non-globular) hydrodynamicbehaviour on chromatography.

These results demonstrate that in physiological ionic strength and pH,i.e. 150 mM PBS pH7.4, at a concentration of 0.5 mg/ml,LD78(Glu28;Glu47) exists as a single, defined, dimeric species with nolarge multimers apparently present, under the methods of analysis used.

Example 127

Mutation of residue Arg47 to Glu prevents the formation of highmolecular weight LD78 multimeric complexes

As detailed in Table 1, pure LD78(Glu47 (mutant 15 of Example 11))protein has been studied in 150 mM PBS pH7.4 buffer using Size ExclusionChromatography on SUPERDEX 75™ resin and by Sedimentation Equilibriumwith wild type LD78 for comparison. The SEC profile of this LD78 mutantis a single, symmetrical peak demonstrating a defined, homogenouspopulation of 21 kDa mass. Analysis of the sedimentation equilibriumdata shows that LD78(Glu47) exists as a monodisperse population ofprotein species with a mass (M.sup.∘_(w)) of 17 kDa corresponding todimers.

The 21 kDa mass measured by SEC in 150 mM PBS pH7.4 is close to thatexpected for an LD78 trimer. Our model of association for this moleculedoes not predict formation of this mass species. The absolute massdetermination by sedimentation equilibrium in this buffer gives veryprecise data proving the molecule is a single molecular species of 15.2kDa. The anomalously high mass by SEC arises due to the asymmetricalshape of the LD78 dimer causing non-ideal (non-globular) hydrodynamicbehaviour on chromatography.

These results demonstrate that in physiological ionic strength and pH,i.e.150 mM PBS pH7.4, at a concentration of 0.5 mg/ml, LD78(Glu47)exists as a single, defined, dimeric species with no large multimersapparently present under conditions of analysis used.

Example 128

Mutation of residues Lys44 to Glu and Arg 45 to Gln prevents theformation of high molecular weight LD78 multimeric complexes

As detailed in Table 1, pure LD78(Glu44; Gln45, (mutant 2 of Example 2))protein has been studied in 150 mM PBS pH7.4 buffer using Size ExclusionChromatography on SUPERDEX 75™ resin and by Sedimentation Equilibriumwith wild type LD78 for comparison. The SEC profile of this LD78 variantis a single peak, FIG. 14, demonstrating a defined population of mass 21kDa. Analysis of the sedimentation equilibrium data shows thatLD78(Glu44; Gln45) exists as a monodisperse population of proteinspecies with a mass (M.sup.∘_(w)) of 16.5 kDa corresponding to dimers.

The 21 kDa mass measured by SEC in 150 mM PBS pH7.4 is close to thatexpected for an LD78 trimer. The model of association for this moleculedoes not predict formation of this mass species. The absolute massdetermination by sedimentation equilibrium in this buffer gives veryprecise data proving the molecule is a single molecular species of 16.5kDa. The anomalously high mass by SEC arises due to the asymmetricalshape of the LD78 dimer causing non-ideal (non-globular) hydrodynamicbehaviour on chromatography.

These results demonstrate that in physiological ionic strength and pH,i.e. 150 mM PBS pH7.4, at a concentration of 0.5 mg/ml, LD78(Glu44;Gln45) exists as a single, defined, dimeric species with no largemultimers apparently present under the conditions of analysis used.

Example 129

Mutation of residue Arg17 to Ser disrupts formation of high molecularweight LD78 multimeric complexes

As detailed in Table 1, pure LD78(Ser17, (mutant 5 of Example 5))protein has been studied at 0.5 mg/ml in 150 mM PBS pH 7.4 buffer, usingSize Exclusion Chromatography on SUPERDEX 75™ resin and by SedimentationEquilibrium with wild type LD78 for comparison. The size exclusionprofile is a single peak of mass 29 kDa. The tetrameric LD78 molecule isexpected to be symmetrical and globular, and, therefore, shouldchromatograph correctly. The observed elution of this LD78 mutantcorrelates with the tetramer species. Analysis of the sedimentationequilibrium data reveals the presence of mixed molecular weight speciesranging from 30 kDa (tetramer) to 100 kDa (dodecamer). No masses higherthan a dodecamer are observed.

These results demonstrate that mutation of Arg17>Ser in LD78 gives amolecule that is incapable of associating to heterogenous high molecularweight complexes. The mutation does not completely block association oftetramers to dodecamers, however, it would appear to energeticallyfavour the equilibrium shifting to the tetramer.

Example 130

Mutation of residue Gln18 to Glu disrupts association of high molecularweight LD78 multimeric complexes

As detailed in Table 1, pure LD78(Glu18, (mutant 29 of Example 19))protein has been studied in 150 mM PBS pH 7.4 buffer, using SizeExclusion Chromatography on SUPERDEX 75™ resin and by SedimentationEquilibrium with wild type LD78 for comparison. The size exclusionprofile is a large broad peak, of mass centred at 48.5 kDa with asmaller component at 160 kDa. The tetrameric LD78 molecule is expectedto be symmetrical and globular, and. therefore, should chromatographcorrectly. The observed elution of this LD78 mutant is anomalously highto correlate with the tetramer species. Analysis of the sedimentationequilibrium data reveals the presence of mixed molecular weight speciesranging from 75 kDa to 170 kDa.

These results demonstrate that mutation of Gln-18>Glu in LD78 gives amolecule that still has some ability to form heterogenous high molecularweight complexes at physiological ionic strength. It is clear from theresults, however, that the mutation does have some disruptive effect onthe association equilibrium. The mass range observed for this mutant issmaller than that seen for wild type LD78 (Table 1), and the SEC profiledemonstrates that the molecule has significantly smaller solution mass.Whilst the mutation of Gln18>Glu does not completely stop the formationof high molecular weight LD78 multimers, it is obvious from thebehaviour of the mutant molecule that this residue plays some role instabilising the multimeric complexes. At protein concentrations <0.5mg/ml in physiological ionic strength, this mutant may well exist as asmaller defined mass species.

Example 131

Mutation of residues Arg17 to Ser and Gln 18 to Glu disrupts formationof high molecular weight LD78 complexes

As detailed in Table 1, pure LD78(Ser17; Glu18, (mutant 30 of Example20)) protein has been studied in 150 mM PBS pH 7.4 buffer, using SizeExclusion Chromatography on SUPERDEX 75™ resin and by SedimentationEquilibrium with wild type LD78 for comparison. The size exclusionprofile is a single peak of mass 25 kDa. The tetrameric LD78 molecule isexpected to be symmetrical and globular, and, therefore, shouldchromatograph correctly. The observed elution of this LD78 mutant isslightly lower than expected for the tetramer species, however, theshift in molecular mass compared to wild type is marked. Analysis of thesedimentation equilibrium data reveals the presence of mixed molecularweight species ranging from 37 kDa to 50 kDa. No masses higher than 50kDa are observed.

These results demonstrate that the combined mutation of Arg17>Ser andGln18>Glu in LD78 gives a molecule that is incapable of associating (at0.5 mg/ml in physiological ionic strength) to high molecular weightcomplexes. In fact the LD78 mutant does not appear to form any molecularweight species higher than 50 kDa.

It is suggested that the results reflect the LD78 mutant exists as atetramer, though some unstable, limited associations can occur.

Example 132

Mutation of residue Phe12 to Gln disrupts formation of high molecularweight LD78 multimeric complexes

As detailed in Table 1, pure LD78(Gln12, (mutant 11 of Example 8))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer usingSize Exclusion Chromatography on SUPERDEX™ 75 and by sedimentationequilibrium with wild type LD78 for comparison. The observed SEC elutionfor this molecule is a single peak of 12.8 kDa, the smallest mass yetseen for an LD78 mutant. Given the non-ideal behaviour of dimeric LD78on SEC (e.g. Example 126), this profile suggests that the mutant existsas a monomer. The sedimentation equilibrium, however, indicates that theLD78(Gln12) is a dodecamer in solution. The original native gel screen(Example 125) showed the presence of a small species. The data in Table1 may reflect an equilibrium between a dodecamer and a smaller speciesand the presence of the sephadex resin may have some physical effect onthe actual equilibrium. Alternatively, the protein may adhere to theresin during chromatography and elute much later with an apparentsmaller mass.

Despite the anomaly in mass determination for this mutant, it is obviousthat mutation of Phe12>Gln gives a LD78 variant that does notmultimerise to the same extent as wild type. The N-terminal region ofthis molecule may play a role in stabilizing more than one state on theequilibrium association pathway.

Example 133

Mutation of Asp26 to Ala disrupts formation of high molecular weightLD78 multimeric complexes

As detailed in Table 1 pure LD78 (Ala26, (mutant 10 of Example 7)) hasbeen studied at 0.5 mg/ml in 150 mM PBS pH7.4 using Size ExclusionChromatography on SUPERDEX™ 75 resin and sedimentation equilibrium withwild type LD78 for comparison. The elution profile gives a single peakof mass 24.5 kDa. Analysis of the sedimentation equilibrium datademonstrates that the protein exists as a monodisperse mass populationwith M.sup.∘_(w) =30 kDa.

This result demonstrates that mutation of Asp26 to Ala gives an LD78molecule which exists at physiological ionic strength as a homogeneoustetramer.

Example 134

Mutation of Glu29 to Arg disrupts association of LD78 dimers to formtetramers

As detailed in Table 1, pure LD78 (Arg29, (mutant 28 of Example 18)) hasbeen studied at 0.5 mg/ml in 150 mM PBS pH7.4 using Size ExclusionChromatography on SUPERDEX™ 75 resin. The elution profile gives a singlepeak of mass 24 kDa. As discussed in Example 86, this observed mass mostprobably relates to a homogeneous dimeric species.

This result demonstrates that mutation of Glu29 to Arg gives an LD78molecule which exists at physiological ionic strength as a singledimeric species.

Example 135

Mutation of Gln 48 to Glu does not affect the multimerisation propertiesof LD78

As detailed in Table 1, pure LD78 (Glu48, (mutant 1 of Example 1))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer usingSize Exclusion Chromatography on SUPERDEX™ 75 and by sedimentationequilibrium with wild type LD78 for comparison. The observed SEC elutionfor this molecule is a single, broad, excluded peak of estimated mass131 kDa. The sedimentation equilibrium shows that LD78(Glu48) exists asa heterogenous range of species from 100 kDa to 600 kDa. Even thoughthis mutant was observed to have increased mobility on native PAGE, thetwo independent size analyses confirm that at this concentration inphysiological buffer, the protein has wild type multimerisation.

In actual fact the observed mass ranges at equilibrium in theultracentrifuge appear to show this mutant forms larger more stablemultimers than wild type. In this case, therefore, introduction of anegative charge at this site may have a stabilizing effect.

Example 136

Mutation of Phe28 to Glu and Gln48 to Glu disrupts formation of highmolecular weight LD78 multimers

As detailed in Table 1, pure LD78 (Glu28; Glu48, (mutant 25 of Example15)) has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 using SizeExclusion Chromatography on SUPERDEX™ 75 resin. The elution profileshows a single, broad asymmetric peak at a molecular mass ofapproximately 60 kDa. The broad asymmetry suggests a heterogeneous mixof mass species. The combined mutations produce a variant that hasmarkedly different multimerisation properties to wild type.

Example 137

Mutation of Arg45 to Ser disrupts formation of high molecular weightLD78 multimeric complexes

As detailed in Table 1, pure LD78 (Ser45,(Mutant 43 of Example 54))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer bysedimentation equilibrium with wild type LD78 for comparison. Analysisof the sedimentation equilibrium data demonstrates that the proteinexists as a monodisperse mass population with M.sup.∘_(w) =25 kDa. Thismass is anomalously high for a dimer and low for a tetramer. Given thatstable trimers are unlikely to form, this mass most probably representsa homogeneous tetramer species.

Example 138

Mutation of Lys44 to Ser disrupts formation of high molecular weightLD78 multimeric complexes

As detailed in Table 1, pure LD78 (Ser44,(Mutant 42 of Example 53))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer bysedimentation equilibrium with wild type LD78 for comparison. Analysisof the sedimentation equilibrium data demonstrates that the proteinexists as a polydisperse population of species ranging in mass from35-48 kDa.

This result demonstrates that the mutation of Lys44 to Ser in LD78 givesa molecule that is incapable of associating (at 0.5 mg/ml inphysiological ionic strength) to high molecular weight complexes. Thefact that no molecular weight species higher than 48 kDa is observedsuggests that the mutation destabilizes the association of tetramers toform dodecamers.

This result is very similar to that obtained forLD78(Ser17;Glu18,(mutant 30 of example 20)) described in Example 131. Itis suggested, therefore, that the results reflect the LD78 mutant existsas a tetramer, though some unstable, limited associations between atetramer and dimer (or monomers) can occur.

Example 139

Mutation of Leu65 to Ala stabilizes a homogeneous high molecular weightLD78 multimeric complex

As detailed in Table 1, pure LD78 (Ala65,(Mutant 51 of Example 63))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer bysedimentation equilibrium with wild type LD78 for comparison. Analysisof the sedimentation equilibrium data demonstrates that the proteinexists as a monodisperse mass population with M.sup.∘_(w) =120 kDa.

The mutation of Leu65 to Ala gives an LD78 molecule that can associateto a stable, homogeneous complex at 0.5 mg/ml in physiological ionicstrength. No other molecular weight species are observed under theseconditions, therefore, the wild-type self-association properties of LD78have been modified.

Example 140

Mutation of Glu66 to Ser disrupts formation of high molecular weightLD78 multimeric complexes

As detailed in Table 1, pure LD78 (Ser66,(Mutant 52 of Example 64))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer bysedimentation equilibrium with wild type LD78 for comparison. Analysisof the sedimentation equilibrium data demonstrates that the proteinexists as a monodisperse mass population with M.sup.∘_(w) =27 kDa.

The results demonstrate that in physiological ionic strength and pH,i.e. 150 mM PBS pH7.4, at a concentration of 0.5 mg/ml, LD78(Ser66)exists as a single, defined, tetrameric species with no large multimerspresent.

Example 141

Mutation of Pro7 to Ala promotes formation of heterogeneous highmolecular weight LD78 multimeric complexes

As detailed in Table 1, pure LD78 (Ala7,(Mutant 59 of Example 88))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer bysedimentation equilibrium with wild type for comparison. Analysis of thesedimentation equilibrium data demonstrates that the protein exists as apolydisperse population of mass species ranging from 400-1000 kDa.

The results demonstrate that in physiological ionic strength and pH,i.e. 150 mM PBS pH7.4 at a concentration of 0.5 mg/ml, LD78(Ala7) existsas a heterogeneous range of large multimeric complexes. The mass rangeobserved for these complexes is much greater than normally observed forwild type LD78 under the same conditions (Comparative Example 3).

The mutation of Pro7 to Ala, therefore, promotes the self-associationproperties of LD78 and the results suggest that the N-terminal arm ofthe protein plays a major role in the multimerisation of this molecule.

Example 142

Mutation of Gln 18 to Ser stabilizes a homogeneous high molecular weightLD78 multimeric complex

As detailed in Table 1, pure LD78 (Ser18,(Mutant 64 of Example 35))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer bysedimentation equilibrium with wild type for comparison. Analysis of thesedimentation equilibrium data demonstrates that the protein exists as amonodisperse mass population with M.sup.∘_(w) =200 kDa.

The mutation of Gln18 to Ser gives an LD78 molecule that can associateto a stable, homogeneous complex of 26 monomers at 0.5 mg/ml inphysiological ionic strength. No other molecular weight species areobserved under these conditions, therefore, the wild-typeself-association properties of LD78 have been significantly modified.

Example 143

Mutation of Tyr61 to Ala stabilizes a homogeneous high molecular weightLD78 multimeric complex

As detailed in Table 1, pure LD78 (Ala 61,(Mutant 73 of Example 68))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer bysedimentation equilibrium with wild type for comparison. Analysis of thesedimentation equilibrium data demonstrates that the protein existsessentially as a monodisperse mass population with M.sup.∘_(w) =400 kDathough a slight upward curvature of Ln A vs ζ was evident.

The mutation of Tyr61 to Ala gives an LD78 molecule that can associateto a stable, homogeneous complex at 0.5 mg/ml in physiological ionicstrength. No other molecular weight species are observed under theseconditions, therefore, the wild-type self-association properties of LD78have been significantly modified.

Example 144

Mutation of Ile19 to Ala gives a homogeneous LD78 monomer

As detailed in Table 1, pure LD78 (Ala 19,(Mutant 80 of Example 81))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer bysedimentation equilibrium with wild type for comparison. Analysis of thesedimentation equilibrium data demonstrates that the protein exists as amonodisperse mass population with M.sup.∘_(w) =6 kDa.

The mutation of Ile19 to Ala gives an LD78 molecule that exists as ahomogeneous monomer at 0.5 mg/ml in physiological ionic strength. Noother molecular weight species are observed under these conditions,therefore, the wild-type self-association properties of LD78 have beencompletely inhibited.

Example 145

Mutation of Val39 to Ala gives a homogeneous LD78 monomer

As detailed in Table 1, pure LD78 (Ala 39,(Mutant 91 of Example 84))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer bysedimentation equilibrium with wild type for comparison. Analysis of thesedimentation equilibrium data demonstrates that the protein exists as amonodisperse mass population with M.sup.∘_(w) =8 kDa.

The mutation of Val39 to Ala gives an LD78 molecule that exists as ahomogeneous monomer at 0.5 mg/ml in physiological ionic strength. Noother molecular weight species are observed under these conditions,therefore, the wild-type self-association properties of LD78 have beencompletely inhibited.

Example 146

Mutation of Arg17 to Glu and Gln18 to Glu disrupts formation of highmolecular weight LD78 multimeric complexes

As detailed in Table 1, pure LD78 (Glu17;Glu18,(Mutant 110 of Example42)) protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer bysedimentation equilibrium with wild type for comparison. Analysis of thesedimentation equilibrium data demonstrates that the protein exists as amonodisperse mass population with M.sup.∘_(w) =30 kDa.

This result demonstrates that the combined mutation of Arg17 to Glu andGln18 to Glu in LD78 gives a molecule that is incapable of associating(at 0.5 mg/ml in physiological ionic strength) to multimeric complexesgreater than a tetramer. Comparison with the results obtained for mutant30 described in Example 131 shows that the more radical substitution ofArg17 to Glu combined with Gln18 to Glu completely disrupts the furtherassociation of tetrameric units.

Example 147

Mutation of Phe12 to Ala partially disrupts the multimerisationproperties of LD78

As detailed in Table 1, pure LD78 (Ala12(mutant 77 of Example 29))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer bysedimentation equilibrium with wild type LD78 for comparison. Analysisof the sedimentation equilibrium data demonstrates that the proteinexists as a polydisperse population of species ranging in mass from110-170 kDa.

This result demonstrates that despite an apparent high mobility inNative PAGE, this variant displays only slight differences inself-association compared to wild type LD78 at 0.5 mg/ml inphysiological ionic strength (Table 1 and Comparative Example 3). Thismay reflect a protein concentration dependence of association such thatat the low protein concentrations in Native PAGE the variant exists as asignificantly smaller mass. It is clear from the results obtained forLD78 variants containing substitutions of Phe12 to Gln or Asp (mutants11 and 115 described in Examples 132 and 148 respectively) that radicalmutation is required at this site to prevent formation of high molecularweight multimers at higher protein concentrations.

Example 148

Mutation of Phe12 to Asp prevents formation of high molecular weightLD78 multimeric complexes

As detailed in Table 1, pure LD78 (Asp12(mutant 115 of Example 106))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer bysedimentation equilibrium with wild-type LD78 for comparison. Analysisof the sedimentation equilibrium data demonstrates that the proteinexists as a monodisperse mass population with M.sup.∘_(w) =30 kDa.

The mutation of Phe12 to Asp gives an LD78 molecule that exists as ahomogeneous tetramer at 0.5 mg/ml in physiological ionic strength. Noother molecular weight species are observed under these conditions,therefore, this mutation inhibits the association of tetramers to higherorder structures.

Example 149

Mutation of Ala4 to Glu disrupts formation of high molecular weight LD78multimeric complexes

As detailed in Table 1, pure LD78 (Glu4(mutant 109 of Example 41))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer bysedimentation equilibrium with wild-type LD78 for comparison. Analysisof the sedimentation equilibrium data demonstrates that the proteinexists as a monodisperse mass population with M.sup.∘_(w) =71 kDa.

The mutation of Ala4 to Glu gives an LD78 molecule that associates to astable homogeneous complex of mass 71 kDa at 0.5 mg/ml in physiologicalionic strength. The self-association of LD78 has, therefore, beendramatically reduced by this mutation demonstrating that the N-terminalarm of the protein is directly involved in the multimerisation process.

Example 150

Mutation of Thr15 to Ala does not significantly affect themultimerisation properties of LD78

As detailed in Table 1, pure LD78 (Ala15(mutant 79 of Example 93))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer bysedimentation equilibrium with wild-type LD78 for comparison. Analysisof the sedimentation equilibrium data demonstrates that the proteinexists as a polydisperse population of species ranging in mass fromapproximately 100-250 kDa with M.sup.∘_(w) =200 kDa.

This result demonstrates that the mutation of Thr15 to Ala gives amolecule that has wild-type association properties at 0.5 mg/ml inphysiological ionic strength. The increased mobility observed on NativePAGE may reflect a concentration dependence of self-association withsmaller mass species predominating at the low concentrations loaded ontogels. A more radical substitution to a polar or charged amino acid wouldelucidate this possibility further.

Example 151

Mutation of Lys36 to Glu does not significantly affect themultimerisation properties of LD78

As detailed in Table 1, pure LD78 (Glu36(mutant 126 of Example 117))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer bysedimentation equilibrium with wild-type LD78 for comparison. Analysisof the sedimentation equilibrium data demonstrates that the proteinexists as a polydisperse population of species ranging in mass fromapproximately 100-250 kDa with M.sup.∘_(w) =200 kDa.

This result demonstrates that the mutation of Lys36 to Glu gives amolecule that has wild-type association properties at 0.5 mg/ml inphysiological ionic strength. The increased mobility observed on NativePAGE may reflect a concentration dependence of self-association withsmaller mass species predominating at the low concentrations loaded ontogels.

Example 152

Mutation of Gln21 to Ser partially disrupts formation of high molecularweight LD78 multimeric complexes

As detailed in Table 1, pure LD78 (Ser21(mutant 81 of Example 86))protein has been studied at 0.5 mg/ml in 150 mM PBS pH7.4 buffer bysedimentation equilibrium with wild-type LD78 for comparison. Analysisof the sedimentation equilibrium data demonstrates that the proteinexists as a monodisperse mass population with M.sup.∘_(w) =112 kDa.

The mutation of Gln21 to Ser gives an LD78 molecule that can associateto a stable homogeneous complex of mass 112 kDa at 0.5 mg/ml inphysiological ionic strength. No other molecular weight species areobserved under these conditions, therefore, the wild-type selfassociation properties of LD78 have been modified.

Example 153

Further LD78 molecules containing amino-acid substitutions which showhigher mobility than wild-type in Native PAGE

In the Native PAGE screening assay described in Example 125, a number ofother LD78 variants have been identified as a single species withgreatly increased mobility than wild type. These variants are: LD78(Glu28,(mutant 17 of Example 13)), LD78 (Asn24,(mutant 24 of Example14)), LD78 (Gln26,(mutant 125 of Example 116)), LD78 (Glu44,(mutant 127of Example 118)), LD78 (Glu45,(mutant 128 of Example 119)) and LD78(Gln66,(mutant 130 of Example 121)).

In addition four variants have been identified which exhibit a mixtureof high and low molecular weight species on Native PAGE. These variantsare: LD78 (Ala43(mutant 69 of Example 87)), LD78 (Ser48(mutant 70 ofExample 27)), LD78 (Ser51(mutant 97 of Example 99)) and LD78(Ala58(mutant 100 of Example 102)).

A further 5 variants have been observed to run with a slight increase inmobility compared to wild-type on Native PAGE which suggests that thoughlarge, they also reflect in a modification of the LD78 self-associationproperties. This conclusion is supported by the analysis of mutants 10,51, 52 and 64 (of Examples 133, 139, 140 & 142 respectively) whichshowed only slight increases in gel mobility and very strikingdifference to wild type when examined by sedimentation equilibrium.These variants are: LD78 (Ser26(mutant 39 of Example 28)), LD78(Ala13(mutant 62 of Example 74)), LD78 (Ala23(mutant 83 of Example 52)),LD78 (Ala32(mutant 88 of Example 70)) and (Ala49(mutant 96 of Example98)).

Some of the variants identified above contain substitutions at sitesknown to be involved in multimerisation described in the previousExamples and it is not unexpected, therefore, to observe changes in gelmobility. The remaining sites, however, are, subject to the limitationsof the gel screen, likely to be involved either directly or indirectlywith the LD78 self-association process.

Example 154

Known natural variants of LD78 exhibit the same multimerisationproperties as the recombinant wild type molecule

As detailed in Table 1, the natural variants LD78 (Leu-Ser-Ala; Pro1;Ser38; Gly46, (mutant 35 of Example 25)) and LD78 (Ala; Pro1, (mutant 34of Example 24)) have been studied by Size Exclusion Chromatography usinga SUPERDEX™ 75 resin and by sedimentation equilibrium with wild type asa control. Both variants are excluded from the SEC resin and thesedimentation equilibrium data shows that they exist as polydispersemasses of 90-200 kDa and 230-350 kDa respectively. The N-terminalextensions and amino acid substitutions in these variants do not,therefore, disrupt the multimerisation properties of the LD78 protein.

Example 155

Definition of the molecular faces involved in LD78 association

Residues important for Monomer→Dimer association

From the examples above, it can be seen that at least residues 19 and 39are important for monomer association to dimers.

Residues important for the Dimer→Tetramer association

From the examples outlined above, two distinct regions of sequence areidentified as important for the stable association of dimers to formtetramers.

(i) Individual mutation of residues Phe28>Glu and Glu29>Arg gives riseto a homogeneous population of dimeric LD78. It is clear, therefore,that residues projecting away from the face of the dimer on strand 1 &1' of the beta sheet form key non-covalent, inter-molecular bonds in thetetramer interface.

(ii) Mutation individually of Arg47>Glu or a combination of Lys44>Glu &Arg45>Gln produces stable LD78 dimers in the absence of higher molecularweight forms. The sequence of residues 43-47 in the turn region linkingstrands 2 (2') and 3 (3') of the beta sheet is, therefore, key for theassociation of LD78 dimers to form tetramers.

Residues important for tetramer→dodecamer and dodecamer→higher ordermultimer

Mutations of Arg17>Glu, Gln18>Glu, Phe12>Asp, Asp26>Ala, Glu66>Ser andAla4>Glu appear to disrupt the tetramer to dodecamer association.Mutations of Gln21>Ser, Leu65>Ala and Phe12>Gln appear to disrupt thedodecamer to multimer association.

Current evidence suggests that these interfaces may overlap or be oneand the same. Mutation of Arg17>Ser and Gln18>Glu individually or incombination appear to disrupt both of the associations outlined above.In this case it is predicted that the sequence region involving residues16-21 on the N-terminal side of strand 1 (1') of the beta sheet are keyfor the associations.

The data so far generated for mutation at residue 48 is ambiguous;however, it may be that this residue also plays a role in the higherorder association of the LD78 molecule.

Example 156

S. cerevisiae Batch fermentation of wild-type LD78, LD78 (Glu44, Gln45)and LD78 (Glu47).

A convenient way of assessing the potential of a transformedSaccharomyces cerevisiae strain to produce recombinant protein is to usea batch fermentation process. This process relies upon providing acontrolled environment in which all of the essential growth nutrientsare present in the medium prior to inoculation. Once inoculation hasoccurred the culture is maintained in an environment appropriate forrecombinant protein expression. Scale-up of recombinant proteinexpression from shake-flask cultures is achieved using fermentercultures. The Saccharomyces cerevisiae strain used for recombinant geneexpression in fermenters is MC2 (see Preparation 3). This strain wasisolated from a chemostat culture of S. cerevisiae strain BJ2168 (seePreparation 3) where galactose was used as a sole and limiting carbonsource. Unlike BJ2168, MC2 exhibits a wild-type phenotype when growingon galactose as a sole carbon source. The batch and fed-batchfermentation strategies developed are designed to complement thisphenotypic characteristic.

The transformed strains used for this example were prepared aspreviously described in Preparation 2. The plasmids used to expressdifferent forms of the molecule are described in Preparation 3(wild-type LD78), Example 2 (Lys44>Glu; Arg45>Gln) and Example 11(Arg47>Glu).

Method

A 1 ml glycerol stock culture (stored at -70° C. in 20% glycerol) wasthawed and used to inoculate 50 ml of sc/glc medium (6.7 g/L yeastnitrogen base w/o amino acids, 10 g/L glucose and 20 ml/L amino acidsolution containing 1 g adenine, 1 g arginine, 5 g aspartic acid, 5 gglutamic acid, 1 g histidine, 15 g iso-leucine, 1.5 g lysine, 1 gmethionine, 2.5 g phenylalanine, 17.5 g serine, 10 g threonine, 2 gtryptophan, 1.5 g tyrosine, 1 g uracil, 6.7 g valine). The culture wasincubated at 30° C. for 24 hrs on a shaking platform after which time 5ml was aseptically removed and used to seed 500 ml of the same sc/glcmedium. This culture was incubated at 30° C. on a shaking platform for24 hr at which point it was used to seed a fermenter (prepared asbelow).

A 5 L fermenter (LSL Biolafitte) was filled with 3.5 L of the definedmedium NAMC#4 containing 41 g ammonium sulphate ((NH₄)₂ SO₄), 5.25 gpotassium dihydrogen orthophosphate (KH₂ PO₄), 2.85 g magnesium sulphate(MgSO₄.7H₂ O), 55 mg calcium chloride (CaCl₂), 16 mg manganous sulphate(MnSO₄.4H₂ O), 18.5 mg copper sulphate (CuSO₄.5H₂ O), 5 mg zinc sulphate(ZnSO₄.7H₂ O), 1 mg potassium iodide (KI), 9 mg sodium molybdate (Na₂MoO₄.2H₂ O), 4 mg ferric chloride (FeCl₃.6H₂ O) and 2 ml PPG₂₀₀₀(antifoam agent). Once sterilised the fermenter was allowed to cool andthe medium within the fermenter was completed with the aseptic additionof 400 ml of a filter sterilised sugar/vitamin concentrate containing 15g glucose, 100 g galactose, 10 mg biotin, 63 mg calcium pantothenate, 63mg pyridoxine hydrochloride, 50 mg thiamine, 50 mg nicotinic acid, 4 mgp-amino benzoic acid, 100 mg myo-inositiol together with 100 ml of afilter sterilised amino acid stock (as for seed culture).

Once the medium was complete the fermenter was set up to maintain thefollowing environment; temperature--30° C., pH--5.0 (using 3M sodiumhydroxide and 3M phosphoric acid as titrants), impeller--750 rpm, airflow rate--2.5 L/min and dissolved oxygen tension above 40% saturation(using increasing impeller rates). After obtaining the runningconditions the fermenter was inoculated with the seed culture (describedpreviously) and the running conditions were maintained for 65 hrs. Atthis point the cell density of the culture was quantified using aspectrophotometer (A₆₀₀) and LD78 levels assessed (in the culturesupernatant) using reverse phase HPLC (Comparative Example 3) using astandard curve of LD78 concentration to give peak height/area.

Using the batch protocol the following data have been

    ______________________________________                                      Specific                Final Biomass                           Final LD78 Productivity    LD78 Species                Level (OD.sub.600)                           Level (mg/L)                                      (wild-type = 1)    ______________________________________    Wild-type   26.6       7          1    LD78        24.3       41         6.4    (Lys44 > Glu;    Arg45 > Gln)    LD78(Arg47 > Glu)                26.0       27         3.9    ______________________________________

Higher productivities from strains expressing disaggregated variants ofLD78 is clearly demonstrated in this experiment.

Example 157

S. cerevisiae Fed-batch fermentation of wild-type LD78 and LD78 variants

The fed-batch strategy is an adaptation of the batch process whichpromotes higher cell densities within the fermentation culture and thusincreases the volumetric level of the recombinant protein. The strainsused for the expression of wild-type and mutant LD78 species were as inExample 156. Transformants were produced as described in Preparation 2and the plasmids used are described in Preparation 2 (wild-type),Example 2 (Lys44>Glu; Arg45>Gln), Example 16 (Phe28>Glu; Arg47>Glu),Example 53 (Lys44>Ser), Example 64 (Glu66>Ser), Example 42 (Arg17>Glu;Gln18>Glu) and Example 19 (Gln18>Glu).

Method

A fermenter was set up as in Example 156. At 18 hr post inoculation afeed was applied to the culture. The feed was 1 L in volume andconsisted of 300 g galactose, 8.3 g ammonium sulphate ((NH₄)₂ SO₄), 1.05g potassium dihydrogen orthophosphate (KH₂ PO₄), 0.57 g magnesiumsulphate (MgSO₄.7H₂ O), 11 mg calcium chloride (CaCl₂), 3 mg manganoussulphate (MnSO₄.4H₂ O), 4 mg copper sulphate (CuSO₄.5H₂ O), 1 mg zincsulphate (ZnSO₄.7H₂ O), 0.2 mg potassium iodide (KI), 2 mg sodiummolybdate (Na₂ MoO₄.2H₂ O), 1 mg ferric chloride (FeCl₃.6H₂ O), 4 mgboric acid (H₃ BO₃), 2 mg biotin, 12 mg calcium pantothenate, 12 mgpyridoxine hydrochloride, 10 mg thiamine, 10 mg nicotinic acid, 1 mgp-amino benzoic acid, 20 mg myo-inositiol in addition to 100 ml of theamino acid stock used in the seed stage. The feed was pumped into thevessel at a rate of 0.22 ml/min. After 48 hrs the feed was stopped and a250 ml pulse of constituents (same as the feed without galactose) wasbatched into the vessel. This batch phase was then allowed to continuefor a further 36 hr after which time the cell density and culturesupernatant were assayed as before (Example 156).

Using transformed MC2 cells and the fed-batch protocol described abovethe following data have been

    __________________________________________________________________________                             Specific                                    Multimeri-            Mutant                Final Biomass                       Final LD78                             Productivity                                    sation    LD78 Species            No  Level (OD.sub.600)                       Level (mg/L)                             (wild-type = 1)                                    Status    __________________________________________________________________________    Wild-type LD78            0   45.3   20    1      Wt    LD78    2   45.8   108   5.3    Dimer    (Lys44 > Glu;    Arg45 > Gln)    LD78    26  50.4   120   5.4    Dimer    (Phe28 > Glu;    Arg47 > Glu)    LD78    42  43.5   50    2.5    Tetramer/    (Lys44 > Ser)                   Dodecamer    LD78    52  39.7   50    2.5    Tetramer    (Glu66 > Ser)    LD78    110 35.3   100   5.0    Tetramer    (Arg17 > Glu;    Glu18 > Glu)    LD78    29  39.3   40    2.0    Dodecamer    (Gln18 > Glu)    __________________________________________________________________________

Higher productivities from strains expressing demultimerised variants ofLD78 is clearly demonstrated in this experiment.

Example 158

Construction of a Pichia Pastoris expression vector for Human LD78

The methylotrophic yeast Pichia pastoris has been used for theproduction of several proteins. High level expression has been obtainedfor a number of proteins in this host but some proteins prove difficultto produce. There is no obvious correlation between the properties of aparticular polypeptide and its ability to be highly expressed in thePichia system. The Pichia pastoris expression system has particularadvantages in its ease of scalability for large scale production.Expression of LD78 was investigated in the Pichia host strain GS115(obtainable from the Phillips Petroleum Company, Bartlesville, Okla.,USA).

The pSW6-LD78 plasmid was used as a source of the α-factor LD78 fusionfor cloning into the P. pastoris expression vector pHILD4. Theexpression vector pHILD4 is a shuttle vector capable of propagation inE. coli and the methylotrophic yeast P. pastoris. The vector comprisessequences derived from the E. coli vector pBR322 and sequences derivedfrom the genome of P. pastoris. The essential features of the vector arethe 5' region of the Pichia AOX1 gene including the regulatable AOX1promoter for high level transcription, the 3' region from the AOX1 genecontaining the transcriptional terminator, a further region from the 3'AOX1 gene which is included together with the 5' AOX1 region to enablesite directed integration of the expression cassette into the hostgenome. The P. pastoris histidinol dehydrogenase gene HIS4 is carriedand used to complement the defective his4 gene in Pichia host strains.The ampicillin resistance gene is carried to allow selection in E. colihosts during genetic manipulation. This vector is similar to the pHILD1vector described in Example 159 except that it also contains a kanamycinresistance cassette which enables selection for multicopy integrantswhen the vector is introduced into Pichia host strains. The pHILD4vector is illustrated in FIG. 15a. Genes for expression may be clonedinto the EcoR1 expression cloning site of the pHILD4 vector. pHILD4 canbe obtained under licence from Phillips Petroleum Company, Bartlesville,Okla., USA.

The pSW6-LD78 vector of preparation 2 was used as a source of thewild-type LD78 gene fused to the Saccharomyces mating type α factorpre-pro leader sequence. Sequences encoding this fusion may be isolatedfrom pSW6-LD78 as a linear DNA fragment following digestion with BglII &BamHI restriction endonucleases. To render the ends of this linearfragment compatible with cloning into the EcoR1 expression cloning siteof the pHILD4 vector it was first necessary to fill in the singlestranded overhangs which result from the BglII/BamHI digestions. Thiswas achieved using the Klenow fragment of E. coli DNA polymerase Itogether with the required deoxynucleoside triphosphates according tostandard methodology. The resultant flush ended fragment was then clonedinto the pHILD4 vector that had been treated with EcoR1 and then bluntended as above. The integrity of the resultant plasmid pLH12 was checkedby a combination of restriction digestion and sequence analysis.

Expression host strains containing pLH12 were constructed using themethod described in Example 159 below.

Example 159

Construction of an improved Pichia Pastoris expression vector forvariants of Human LD78

Whilst pLH12 of Example 158 was used for the early expression analysis,this vector was improved upon as shown in this example and the resultantimproved vector pLH23 was used for the construction of Pichia expressionvectors for LD78 variants. Pichia expression vector pHILD1 (FIG. 16) isa shuttle vector capable of propagation in both E. coli (for ease ofgenetic manipulation) and in the methylotrophic yeast Pichia pastoris.The S. cerevisiae mating type factor alpha secretion signals wereincorporated into the pHILD1 vector to enable export of the LD78 proteinto the medium. pHILD1 can be obtained under licence from the PhillipsPetroleum Company, Bartlesville Okla., USA. The vector comprisessequences derived from the E. coli vector pBR322 and sequences derivedfrom the genome of Pichia pastoris. The essential features are the 5'region of the Pichia alcohol oxidase (AOX1) gene including theregulatable AOX1 promoter for high level transcription, the 3' regionfrom the AOX1 gene containing the alcohol oxidase transcriptionalterminator sequence, a further region from the 3' part of the AOX1 geneis included which together with the 5' AOX1 region is required forsite-directed integration of the expression cassette into the hostgenome. The P. pastoris histidinol dehydrogenase gene HIS4 is carriedand used to complement the defective his4 gene in Pichia host strains.The ampicillin resistance gene is carried to enable selection in the E.coli hosts used during genetic manipulation. The pHILD1 vector wasmanipulated to allow expression of the synthetic LD78 gene (obtainedfrom pUC18-LD78) of Preparation 1 under the control of the alpha factorsecretion signal. pHILD1 does not carry any sequences encoding secretionsignals to allow export of heterologous proteins. To include such asignal, the vector was manipulated by the addition of sequences from theS. cerevisiae alpha-factor leader. The vector was further engineered toprovide a more optimal promoter context and to remove undesirableHindIII restriction sites which may interfere with the cloning of theLD78 gene from pSW6-LD78 of Preparation 2, a BamHI site was thenintroduced 3' to the remaining HindIII to allow cloning of the LD78 gene(pUC18-LD78 of Preparation 1) on a HindIII-BamHI restriction site and toinclude a kanamycin resistance cassette enabling the selection ofmulticopy integrants in transformed Pichia host strains. The stages ofthe manipulations are below. An outline of the strategy used is shown inFIG. 18.

Inclusion of alpha-factor secretion signals

The alpha-factor sequences were cloned into the pHILD1 vector from theS. cerevisiae expression vector pSW6 (FIG. 2) (see Preparation 2 fordetails). The alpha-factor sequences were isolated from pSW6 on a ca 430bp BglII-BamHI DNA fragment, this fragment contains the alpha-factorsequences fused to a human epidermal growth factor synthetic gene (EGF).The overhanging ends of this DNA fragment were first filled in usingklenow fragment of E. coli DNA polymerase I together with the requireddeoxynucleoside triphosphates according to standard methodology. Theflush-ended fragment was then cloned into the pHILD1 vector that hadbeen treated with EcoRI and then blunt-ended as above. The integrity ofthe resultant plasmid pLH001 was checked by a combination of restrictiondigestion and DNA sequence analysis. The primer use for sequenceanalysis was BB5769 (5'-GCATTCTGACATCCTCT-3' SEQ ID NO 168). Thesequence of the α factor coding sequence was confirmed.

Mutagenesis to optimise vector for variant LD78 expression

The pLH001 vector was further modified to remove unwanted HindIIIrestriction sites, to optimise the promoter region and to introduce aBamHI site. Relevant fragments were cloned into a bacteriophage M13vector for site-directed mutagenesis. The fragments cloned, the primersused for mutagenesis, and the primers used for sequencing are detailedbelow. Furthermore, a kanamycin resistance cassette was modified forintroduction into the final expression vector to allow selection formulticopy integrants when the vector is introduced into Pichia hoststrains.

A ca 1220 bp SacI-SacI fragment was isolated from pLH001 and cloned intoM13 mp19. This M13 construct was then used for mutagenesis in which aHindII site was removed using oligonucleotide primer BB6040(5'-CGTTAAAATCAACAACTTGTCAATTGGAACC-3' SEQ ID NO 169), the mutants wereidentified by sequence analysis with sequencing primer BB6296(5'-GGAAATCTCACAGATCT-3' SEQ ID NO 170). This fragment was furthermodified by deletion mutagenesis to optimise the 5' untranslated leaderregion preceding the AOX1 promoter,which is now identical to that foundin the natural 5' untranslated leader of the AOX1 gene on the Pichiagenome. Having the correct context around the 5' untranslated leader ispreferred for maximal expression. The mutagenesis primer used for thisstep was BB8461 (5'GAAGGAAATCTCATCGTTTGAATA-3' SEQ ID NO 171). Themutant was identified by sequence analysis with sequencing primer BB8740(5'-GCTAATGCGGAGGATGC-3' SEQ ID NO 172).

Two further HindIII sites were removed from the ca 770 bp SacI-XbaIfragment of pLH001 by mutagenesis. The SacI-XbaI fragment of pLH001 wasfirst cloned into M13 mp18 and one of the HindIII sites was removedusing the primer BB6394 (5'-CCGGCATTACAACTTATCGATAAGCTTGCAC-3' SEQ ID NO173). The identity of this mutant was confirmed by sequence analysisusing the sequencing primer BB6037 (5'-GCGCATTGTTAGATTTC-3' SEQ ID NO174). A second HindIII site was removed from this newly mutagenisedfragment using mutagenesis primer BB6841 (5'-CTTATCGATCAACTTGCACAAACG-3'SEQ ID NO 175). The correct mutant was identified by sequence analysisusing sequence primer BB6037 (see above).

Before reassembly, a BamHI site was introduced into the HindIII deletedSacI-XbaI fragment to allow subsequent cloning of the LD78 gene ofPreparation 2 on a HindIII-BamHI fragment. The mutagenesis primer usedto introduce the BamHI site was BB6189 (5'GTCATGTCTAAGGCGGATCCTTATTAAC-3' SEQ ID NO 176). The identity of themutant was identified using sequencing primer BB5769(5'-GCATTCTGACATCCTCT-3' SEQ ID NO 168).

Modification of the Kanamycin resistance cassette

A kanamycin resistance cassette was purchased from Pharmacia BiosystemsLimited, Davy Avenue. Knowlhill, Milton Keynes, MK5 8PH. Great Britain.This cassette is supplied as an EcoRI fragment by Pharmacia and this wascloned into M13 mp19 as an EcoRI fragment. The internal HindIIIrestriction site was deleted using mutagenesis primer BB8661(5'-GAGAATGGCAACAACTTATGCATT-3' SEQ ID NO 177). The mutation wasconfirmed using sequencing primer BB6038 (5'-CCAACATCAATACAACC-3' SEQ IDNO 178).

Reassembly of expression vector

The vector was reconstructed in a stepwise manner using the Phillipspetroleum vector pHILD1 as a backbone for the cloning.

To rebuild the expression vector including the mutagenised fragments,the modified ca 770 bp SacI-XbaI fragment was first ligated intoSacd-XbaI treated pHILD1 vector. The integrity of the recombinantconstruct was then confirmed by restriction analysis and DNA sequenceanalysis using the oligonucleotide sequencing primer BB6037(5'-GCGCATTGTTAGATTTC-3' SEQ ID NO 174), the construct was calledintermediary vector 1. The modified SacI-SacI fragment was next clonedinto intermediary vector 1 which had been treated with SacI and calfintestinal phosphatase. The resultant construct, named intermediaryvector 2, was again confirmed by restriction analysis and DNA sequenceanalysis with oligonucleotide primers BB6296 (5'-GGAAATCTCATAGATCT-3'SEQ ID NO 170) to read through the deleted HindlI site and BB8740(5'-GCTAATGCGGAGGATGC-3' SEQ ID NO 172) to read through the optimised 5'untranslated leader region. Intermediary vector 2 is a homologue ofpHILD1 which lacks the unwanted HindIII sites, has an optimised 5'untranslated region, contains sequences encoding the S. cerevisiaealpha-factor secretion signals followed by the remaining HindIII siteand which has a BamHI site 3' to the HindIII site to allow cloning ofthe synthetic LD78 gene described in Preparation 1.

A 1,200 bp HincII fragment containing the mutagenised kanamycin cassettewas removed from the M13 mp19 mutagenesis vector (used to remove theHindIII site from the kanamycin resistance gene) and cloned into theunique NaeI site of the intermediary vector 2. The vector was renamedpLHD4. The integrity of pLHD4 was confirmed by restriction analysis. Amap of pLHD4 is shown in FIG. 15b. pLHD4 contains the human EGF genefused to the S. cerevisiae alpha factor secretion signal.

Construction of the improved LD78 Pichia expression vector

The improved expression vector for wild-type LD78 expression wasconstructed by cloning a HindIII-BamHI fragment of pSW6-LD78(Preparation 2) into pLHD4. (This HindIII-BamHI fragment contains thesynthetic LD78 gene fused to the 3' end of a sequence encoding the 5amino acids of the yeast alpha factor which precede the KEX2 cleavagesite required for liberation of the mature peptide following secretionfrom the Pichia host).

The HindIII-BamHI fragment was obtained by restriction digestion of theS. cerevisiae expression vector pSW6-LD78. This fragment was purified ona 1.5% low melting temperature agarose gel then ligated toHindIII-BamHI, calf intestinal phosphatase treated pLHD4. The resultantrecombinant was called pLH23. The vector is shown in FIG. 17. Theintegrity of the construct was confirmed by restriction analysis andsequencing analysis using the sequencing primer BB5769(5'-GCATTCTGACATCCTCT-3' SEQ ID NO 168). FIG. 18 shows the strategy forthe construction of pLH23.

Example 160

Construction of P. pastoris expression vectors for demultimerised LD78variants.

The improved vector of Example 159 was used as the basic expressionvector for all LD78 variants. The DNA encoding the LD78 variants wasobtained from the S. cerevisiae vector described in Examples 16, 2 ,11,5, 20, 18, 1, 15 and 8. Briefly, the plasmid DNA from these variousexamples was digested with HindIII and BamHI restriction endonucleases.This releases a fragment containing sequences which encode the LD78variant fused to a sequence encoding the last 5 amino-acids of the S.cerevisiae mating factor type alpha. The HindIII/BamHI DNA fragmentswere ligated into HindIII/BanHI-treated pLH23. The resultant vectorstogether with the LD78 variant carried can be seen in the table below

    ______________________________________    Mutant No   Mutation          Pichia Vector    ______________________________________    26          Arg 47 > Glu; Phe28 > Glu                                  pLH    25    2           Lys44 > Glu; Arg45 > Glu 26    15          Arg47 > Glu              27    5           Arg17 > Ser              28    30          Arg17 > Ser; Gln18 > Glu 30    28          Glu29 > Arg              29    1           Gln48 > Glu              31    25          Phe28 > Glu; Gln48 > Glu 24    11          Phe12 > Gln              32    ______________________________________

Expression hosts for these various plasmids were constructed accordingto the method described in Example 161 below.

Example 161

Construction of Pichia expression strains.

pLH12 plasmid DNA prepared as in Example 158 was linearised by cuttingwith the restriction endonuclease Sac I. This was to enable theexpression cassette to integrate via homologous recombination ofsequences on the expression cassette and the host chromosome. Thelinearised plasmid was then transformed into P. pastoris strain GS115(NRRL Y-1585) which has the genotype his4. The use of strain GS115 isnot critical for use either in this preparation or in the invention ingeneral. Any suitable strain can be used, such as, for example, strainKM71 or SMD1163 which have the genotypes his4, AOX1::ARG4 and his4,prB1, pep4 respectively. Strains GS115 and KM71 are described inPhillips patent number AU-B-63882/86. These hosts can be obtained underlicence from the Phillips Petroleum Company, Bartlesville, Okla., USA.

Using the method described below the plasmid DNA was transformed intothe host strain.

Briefly, yeast strain GS115 was grown overnight in 200 mL of YEPD mediumat 30° C. on an orbital shaker. Cultures at an A₆₀₀ of between 0.1 and0.3 were harvested by centrifugation at 300 rpm for 5 mins, washed insterile water, recentrifuged, washed in SED buffer (Appendix A at theend of the Examples), recentrifuged, washed in 1M sorbitol,recentrifuged and resuspended in 20 mL SCE buffer (Appendix A). Cellswere then incubated at 30° C. with the enzyme zymolyase to remove thecell wall. Spheroplasting was allowed to continue until approximately70% of the cells had been turned into spheroplasts. These were thencollected by gentle centrifugation (750 xg 10 mins). Spheroplasts werethen washed in 1M sorbitol and resuspended in 600 μL CAS buffer(Appendix A). 100 μL aliquots of spheroplasts were then incubated for 10mins with 10 μg of the linearised DNA. 1 mL of PEG buffer was then addedand incubated for a further 10 mins. After collecting the spheroplastsby gentle centrifugation and aspirating the PEG solution the cells wereresuspended in 150 μL of SOS medium (Appendix A) and incubated for 20mins. After the addition of 850 μL of 1M sorbitol the cells were readyfor plating on regeneration agarose.

100 μL of transformed spheroplasts were then added to 10 mL of molten(42° C.) agarose-sorbitol regeneration medium and poured ontoagarose-sorbitol base plates and allowed to grow for 5-7 days at 30° C.

All yeast media and transformation buffers were as described in theappendix.

After 5-7 days transformants were collected along with the agaroseoverlay they had been growing in, transferred to a 50 mL centrifuge tubeand resuspended in 50 mM sodium phosphate buffer pH6 and after suitablemixing and agitation to remove the cells from the agarose they werediluted and plated onto YEPD agar plates containing the antibiotic G418at concentrations between 0 and 2,000 μg/mL. Only cells in which severalcopies of the expression cassette had integrated into the hostchromosome would be able to grow on high levels of antibiotic by virtueof their enhanced kanamycin resistance. Such cells are deemed desirableis since they will also be carrying several copies of the LD78 gene.Previous work has shown such multicopy integrants to be high producersunder conditions were the foreign gene is expressed (Clare et al(1991)). Plates were incubated at 30° C. for 5-7 days. Coloniesoccurring on plates containing high concentrations of the antibioticwere then picked and streaked onto fresh MD agar plates. Single colonieswere obtained after 3-4 days growth at 30° C.

In order to determine the number of copies of the expression cassettethat had integrated onto the host chromosome a Southern blottingtechnique based on that described by Clare et al. (1991) was employed.

Briefly, chromosomal DNA was prepared from transformed cells anddigested with the restriction endonuclease BglII. The resulting DNAfragments were separated by gel electrophoresis and transferred tonitrocellulose by electroblotting. The resulting Southern blot was thenincubated with a labelled probe that recognises one of the DNA sequenceson the expression cassette (for example HIS4). The probe will alsorecognise the single copy of the his4 gene present on the hostchromosome. By comparing the intensity of the signal from the knownsingle copy with the unknown multicopy signal (by scanning densitometry)it is possible to quantify the number of copies present.

Exactly the same method was employed for transformation and constructionof expression strains from the LD78 variant expression vectors describedin Example 160.

Example 162

Expression of wt LD78 in Pichia pastoris

Wild-type expression hosts contained pLH12 as described in Example 158.

Single colonies of transformed strains were used to inoculate 5 mL ofBMGC medium (Appendix A) and the cultures were grown overnight at 30° C.on an orbital shaker. This 5 mL overnight culture was then used toinoculate 2 L baffled shake flasks containing 50 mL of the medium BMGC.After 24 h growth at 30° C. on an orbital incubator cells were harvestedby centrifugation at 300 rpm for 5 min and resuspended in 50 mL of BMMC(Appendix A). This induces gene expression from the AOX1 promoter.Induction was carried out by growth in the methanol containing medium at30° C. for 48-72 h.

After either 48 h or 72 h the culture supernatant was collected bycentrifugation at 300 rpm for 5 min to remove cells. This supernatantwas used for further analysis and purification of LD78 according to themethods described in Preparations 3 and 4. Levels of wild-type LD78produced using this method are typically 3-5 mg/L as determined by HPLC.

Such levels can be improved by growing the producing strain in afermenter. A single colony was inoculated into 5 mL of MD medium(Appendix A) and grown overnight at 30° C. in an orbital incubator. Thisculture was then used to inoculate 500 mL of YEPGlycerol medium(Appendix A) in a 2 L baffled flask. This culture was grown for between24-48 h and used as an inoculum for the fermentor. The 5 L fermenter wasautoclaved with 3.5 L of the High Cell Density (HCD) medium (AppendixA). After adjusting the pH to 5.85 with ammonia solution and theaddition of 10 mL of a trace element solution (PTM₁, (Appendix A)) thefermenter was inoculated with the culture described above. Growthconditions are typically pH5.85 (maintained by the addition of ammoniasolution on demand), 29.8° C., 800-1200 rpm, 1-2 vvm air, 20-100% DOT.After 20-24 h the carbon source in the medium was exhausted and amethanol feed (containing 5 mL/L of trace element solution PTM₁ and 2mL/L biotin stock solution--0.2 g/L) started at 3.4 g/h. After 24-30hours the feed rate was increased to 6 g/h for approximately 20 h. Afterthis the feed rate was increased or decreased to keep the residualmethanol concentration in the broth between 1 and 10 g/L (as determinedby gas chromatography). The fermentation was run for between 70-180 hand wild-type LD78 levels in the broth were determined to be 60-100 mg/Lby HPLC.

The material produced using the Pichia expression system was purifiedand characterised using the techniques applied to the material producedby Saccharomyces (see Preparation 3, 4 and 13).

Example 163

Enhanced expression of demultimerised mutants

The expression constructs for the demultimerised variants as describedin Example 160 were introduced into Pichia host strain GS115 accordingto the method of Example 161.

It was generally noted that mutations of the LD78 gene that resulted ina demultimerised form of the molecule gave higher levels of expressioninto culture supernatants than did the wild-type LD78 molecule.

As mentioned in Example 162, the expression level in shake flaskinductions was determined to be 3-5 mg/L for the wild-type LD78molecule. When production strains containing integrated expressioncassettes of demultimerised mutants were grown as detailed in Example157 expression levels were seen to be elevated to the order of 50-200mg/L (specifically mutant 26 (LD78 Glu28, Glu47)--158 mg/L, mutant 2(LD78 Glu44, Gln45)--76 mg/L, mutant 15 (LD78 Glu47)--63 mg/L, mutant 5(LD78 Ser17)--79 mg/L, mutant 30 (LD78 Ser17, Glu18)--138 mg/L, mutant28 (LD78 Arg29)--169 mg/L). This phenomenon was not restricted to thePichia system but was also noted with the Saccharomyces system (seeExamples 156 and 157).

When production strains containing the demultimerised mutant expressioncassettes were grown in fermenters expression levels were againenhanced. Glycerol stock cultures were used to inoculate 500 mL ofYEPGlycerol medium in a 2 L baffled shake flask. This was grown for18-24 h at 30° C. in an orbital shaker. This culture was used as aninoculum for the fermenter. The 5 L fermenter was prepared as detailedin Example 162. After the batch phase carbon was exhausted a limitingglycerol feed (500 g/L glycerol, 5 mL/L trace elements PTM₁, 2 mL/Lbiotin stock solution 0.2 g/L) was started and run for 3-6 h at 14 g/h.Then the glycerol feed rate was reduced to 10 g/h and a methanol feed(methanol plus 5 mL/L trace elements PTM₁ and 2 mL/L biotin stocksolution 0.2 g/L) started at 5 g/h. The methanol feed was increasedexponentially with time to arrive at a final feed rate of 30 g/h after atotal elapsed fermentation time of 75 h. During this period growthconditions were as detailed in Example 162. This process resulted in theproduction of 1.5 g/L of demultimerised mutant 26 (LD78 Glu28, Glu47)into the fermentation broth, compared with 60-100 mg/L of the wild-typeLD78 molecule. Clearly, expression levels may be dependent on the numberof expression cassettes integrated into the host chromosome. In order tocompare expression levels of demultimerised mutants with those of thewild-type, differences in copy number must be taken into account. Thestrain producing wild-type LD78 has 4 copies of the expression cassettecompared to 42 for the mutant 26 producing strain. Even allowing forthis difference, however, mutant 26 is produced at higher than expectedlevels (3.1 mg/L/copy as opposed to 0.75-1.25 mg/L/copy for wild-type).

This phenomenon of enhanced expression of demultimerised mutants in afermenter was also observed with the Saccharomyces system (see Example157).

Example 164

Demultimerised mutants are active in an in vitro receptor binding assay

The effect of the mutations on LD78 biological activity was assessedinitially by measuring their ability to displace radio-labelled LD78from the murine stem cell line FDCP cell mix (A4 cells) (Dexter et al.,J. Exp. Med. 152 1036 (1980)). The A4 FDCP cell mix cell line isavailable on request from the Paterson Cancer Research Institute,Department of Haematology, Wilmslow Road, Manchester, M20 9BX, UnitedKingdom).

The assay procedure is as follows: FDCP-mix A4 cells are diluted withfresh growth medium on the day before use to give 1-2×10⁵ /ml (usually2-4 fold). On the day of the assay, cells are counted and then harvestedby centrifugation. After washing once in serum free medium and once inbinding medium, the cells are resuspended at 5×10⁶ /ml in binding medium(RPMI 1640+20 mM HEPES +1 mg/ml BSA). 200 μl of the cell suspension ispipetted into Eppendorf tubes, followed by 25 μl of unlabelledcompetitor, made up at 10× the required final concentration, and 25 μllabel prepared in the same way. The final concentration of labelledligand used is 0.5 nM, i.e. 3.85 ng/ml. The tubes are incubated on asuspension mixer for 2 hours at room temperature. 1 ml of cold PBS isthen added and the tubes centrifuged at 2000 rpm. After washing in 2further volumes of PBS the cells are finally transferred to vials andthe radioactivity measured by counting using a Packard Cobra Auto-Gammacounter. The assay was performed in triplicate, and the binding of ¹²⁵I-LD78 in the presence of excess cold LD78 or LD78 mutant was comparedto binding in the absence of cold material.

LD78 or LD78 mutant was diluted in binding medium to provide a range ofconcentrations. Routinely, concentrations of 3.85 μg/ml and 0.385 μg/mlwere prepared, which following a ten-fold dilution into the assay,yielded concentrations of cold material that were 100- and 10-fold theconcentration of ¹²⁵ I-LD78 respectively. ¹²⁵ I-LD78 was prepared byAmersham plc.

For more detailed characterisation of selected variants, a range ofsample concentrations from 0.01-100 ng/ml was employed to construct adetailed dose response curve. To ensure comparability between assays,the activity of LD78 variants was expressed as percentage of thewild-type activity based on IC50 values. Wild-type LD78 was alwaysincluded as a control. Thus wild-type activity is represented as 100%; avariant that with an IC50 ten times that of wild-type as 10% (i.e. bindsthe receptor 1/10 as well) and a variant with an IC50 1/2 that ofwild-type as 200% (i.e. binds the receptor twice as well).

The receptor binding data for 53 LD78 variants are shown in Table 2,along with a summary of the relevant physicochemical data relating totheir multimerisation state.

                                      TABLE 2    __________________________________________________________________________    BIOLOGICAL ACTIVITY OF SCI MUTANTS                  Size on    Mutant        Residue   Native                       AUC       Receptor    No. No.  Mutation                  Gel   mean!                           Structure                                 Binding                                      % WT    __________________________________________________________________________    0        LD78 WT   160 Wt    1    100    1   48   Gln > Glu                  Large?                       400 Wt    2    2   44   Lys > Glu                  Small                       16  D     4    5        45   Arg > Gln    5   17   Arg > Ser                  Mixed                       57.5                           T/Do  2    25    10  26   Asp > Ala                  Small                       35  T     1    77.4    11  12   Phe > Gln                  Mixed                       98  T/Do  1    34    26  28   Phe > Glu                  Small                       16  D     4    1        47   Arg > Glu    28  29   Glu > Arg                  Small                       T   2     7.7    29  18   Gln > Glu                  Small                       130 Do    1    30  17   Arg > Ser                  Small                       41  T     3    4.2        18   Gln > Glu    35  -3   >Leu WT   155 Wt    4    3        -2   >Ser        -1   >Ala        1    Ser > Pro        38   Gly > Ser        46   Ser > Gly    37  5    Asp > Ser                  WT       Wt    1    45    38  24   Ile > Ala                  WT       Wt    1    50    40  29   Glu > Ser                  WT             4    42  44   Lys > Ser                  Small                       45  T/Do  1    18    43  45   Arg > Ser                  Small                       25  T     3    45  52   Asp > Ser                  WT             4    48  60   Lys > Ser                  WT       1    52  66   Glu > Ser                  Large                       27  T     1    161.5    54  1    Ser > Ala                  WT             1    145    60  8    Thr > Ala                  WT             1    62  13   Ser > Ala                  Large                       370 Wt    1    66    63  16   Ser > Ala                  WT             3    64  18   Gln > Ser                  Large                       200 Wt    4    66  27   Tyr > Ala                  WT             2    47    68  35   Ser > Ala                  WT       Wt    1    125    70  48   Gln > Ser                  Mixed          2    71  53   Pro > Ala                  WT             3    75  67   Leu > Ala                  WT             1    77  12   Phe > Ala                  Small                       150       3    79  15   Thr > Ala                  Small                       180       1    82  22   Asn > Ser                  WT             1    84  25   Ala > Ser                  WT             1    85  28   Phe > Ala                  WT             3    87  31   Ser > Ala                  WT             3    94  42   Leu > Ala                  WT             1    97  51   Ala > Ser                  Mixed          2    101 26   Asp > Ala                  WT             2        29   Glu > Arg    102 26   Asp > Ala                  Small          4        29   Glu > Arg        47   Arg > Glu    __________________________________________________________________________     KEY: AUC = Analytical Ultra Centrifugation (kDa)     Receptor Binding: 1 = Wildtype     2 = 1/10 to 1/2 Wt     3 = 1/100-1/10 Wt     4 = Inactive     T = Tetramer     T/Do = Tetramer/Dodecamer equilibrium     D = Dimer     WT = Wild type

The following facts emerge from this analysis:

1)The majority of the variants with wild-type or minimally affectedmultimerisation properties exhibit wild-type or close to wild-typereceptor binding.

2)There is a clear subset of variants which, though wild-type withrespect to size, are clearly affected in their ability to compete withwild-type LD78 for receptor binding. The mutations in these variantspresumably define the residues involved in interacting with thereceptor. These key residues include Lys-44, Arg-45, Arg-17, Gln-18,Phe-28 and Glu-29.

3)Most of the de-multimerised variants appear to be compromised inreceptor binding. This implies either that the residues involved inmultimerisation are also involved in receptor binding, or that receptorbinding requires a multimeric form of LD78. Wild-type receptor bindingactivity has not been seen in variants smaller than a tetramer. This issummarised in FIG. 19. The numerals refer to the number of mutants foundin each category. Mutants shown lying between the tetramer and dodecamerpositions represent an equilibrium between the two states.

4)Variants in which the N terminus of LD78 are extended show greatlydiminished ability to compete for receptor binding. Surprisingly, theseinclude the forms of LD78 described previously such as in variant #35(WO-A-9104274) and variant #34 (JP-A-03228683). In contrast, deletion ofN-terminal residues appears to have minimal effect on receptor binding.The other N-terminal form described in the literature (Pragnell et al.,CRC Beatson Laboratories Scientific Report, Beatson Institute for CancerResearch, Glasgow, Scotland) does not express in the yeast expressionsystems described in this application.

5)The residues implicated in receptor binding map to two defined regionson the surface of the LD78 model described above. One region flanks theN-terminal serine and includes residues in the β-turn around residues44-48 (Lys-Arg-Ser-Arg-Gln).

Taken together, these data suggest that the active form of LD78 is atetramer. FIG. 20 shows a view of the model of tetrameric LD78, showingthe dramatic clustering of residues implicated in receptor binding. Inthis model of LD78 structure and function, mutations at the interfacebetween dimers exert their effect on receptor binding indirectly, bydisrupting the formation of the active, tetrameric species. A secondimplication of this model is that the N-terminal extended forms of LD78are probably inactive proforms of the molecule, at least as regards thereceptor present on A4 cells.

Both of these conclusions are surprising in view of the prior art. InWO-A-9104274 the N-termini of the LD78 forms they describe was notdefined. The material was apparently biologically active, perhaps as aresult of processing by proteases present in their in vitro assay ofcolony formation, or in view of the high concentrations of materialused.

Although the active species of the SCI family of molecules has been amatter for speculation, it was recently asserted that for LD78 theactive species is a monomer (Mantel et al., (1992), loc. cit.). This wasbased on the observation that E.coli-derived LD78, disaggregated in 30%acetonitrile & 0.1% TFA, was 1000-fold more active in various in vitrocolony forming unit assays on the haematosis lineage precursors BFU-Eand GM-CFC cells. We can only speculate that this large differencereflects a problem with the activity of the aggregated E.coli derivedmaterial.

Example 165

Demultimerised mutants can inhibit the proliferation of haemopoieticprogenitor cells (Day 12 CFU-S)

The ability of mutant #10 (Example 7) to inhibit the formation of murineday 12 CFU-S cell colonies was measured in vitro according to thefollowing method. The activity was compared to that of mutant #82(Example 94), which is wild-type with respect to structure and receptorbinding.

Day 12 CFU-S cells were sorted from normal murine bone marrow cells asdescribed in Lord and Spooncer (1986) Lymphokine Research 5:59-72.Sorted cells (between 500-1000) were plated in soft agar and assayed fortheir colony forming ability according to the method described inHeyworth and Spooncer (1992) in "Haemopoiesis--A Practical Approach"page 37 IRL Press (Testa and Molineux, Eds).

Growth factors were supplied from conditioned medium of L cells andAF1-19T cells. Each of the conditioned media was used at 10% asdescribed in Pragnell et al., (1988) Blood 72:196-201. LD78 mutant 10 or82 was added at 150 ng/ml, 15 ng/ml, 1.5 ng/ml or 0.15 ng/ml to the topagar in 10 μl of PBS and allowed to diffuse through teh plate. Theplates were then incubated at 37° C. in 5% O₂, 5% CO₂ for 14 days.Colonies were counted with an inverted microscope. All assays were runin triplicate. 150 ng/ml of LD78 wild type protein of Preparations 1 to4 and PBS were used as controls in this experiment.

Results were expressed as a percentage of the control treted withcarrier PBS alone. The Mutant 10 used in this assay will inhibit colonyformation fo day 12 CFU-S cells at concentrations down to 1.5 ng/ml.Both mutant 10 (FIG. 24) and 82 (FIG. 25) show similar potency withoptimum inhibitors at 15 ng/ml. This shows that a demultimerised variantcan exert functional effects as well as binding to the receptor.

                  Appendix A    ______________________________________    Media recipes    BMGC    Quantities per liter:    Sodium phosphate buffer 1M, pH6                       100 mL    Casamino acids (100 g/L)                       100 mL    Yeast Nitrogen Base (13.4 g/L)                       100 mL    Biotin (0.2 g/L)   2 mL    Glycerol           10 mL    Filter sterilise    BMMC    As above but replace glycerol with 5 mL of methanol.    YEPD    Yeast extract      10 g/L    Peptone            20 g/L    Glucose            10 g/L    For solid medium add 15 g/L agar    Autoclave at 121° C. 15 mins    YEPGlycerol    As above but replace glucose with glycerol    HCD    H.sub.3 PO.sub.4(85%)                       21 mL/L    CaSO.sub.4.H.sub.2 O                       0.9 g/L    K.sub.2 SO.sub.4   14.28 g/L    MgSO.sub.4.7H.sub.2 O                       11.7 g/L    KOH                3.9 g/L    Glycerol           50 g/L    pH is about 1.7 when made up. Bring pH to 4 in the fermentor    with ammonia solution (prior to sterilization). Sterilize in the    fermentor and bring pH to 5.85 with ammonia solution prior to    inoculation.    To the 3.5 L of medium in the fermentor add 10 mL of the following    trace element solution (PTM.sub.1)    CuSO.sub.4.5H.sub.2 0                       6 g/L    KI                 0.8 g/L    MnSO.sub.4.H.sub.2 O                       3.0 g/L    NaMoO.sub.4.2H.sub.2 O                       0.2 g/L    H.sub.3 BO.sub.3   0.02 g/L    CoCl.sub.2.6H.sub.2 O                       0.5 g/L    ZnSO.sub.4         20 g/L    H.sub.2 SO.sub.4   5 mL/L    FeSO.sub.4.7H.sub.2 0                       65 g/L    Biotin             0.2 g/L    MD    Yeast nitrogen base                       13.4 g/L    Biotin             0.4 g/L    Glucose            20 g/L    Filter sterilise    For solid medium add 15 g/L agar    Transformation buffers and reagents    SED    Sorbitol           1M    EDTA (pH8)         25 mM    DTT                50 mM (add just prior to use)    SCE    Sorbitol           1M    EDTA               1 mM    Sodium citrate buffer pH5.8                       10 mM    CAS    Sorbitol           1M    Tris-Cl pH7.5      10 mM    CaCl.sub.2         10 mM    PEG solution    PEG 3350           200 g/L    Tris-Cl pH7.5      10 mM    CaCl.sub.2         10 mM    Prepare fresh and filter sterilise. Discard if pH is below 7.    SOS    Sorbitol           1M    YEPD               x0.3    CaCl.sub.2         10 mM    Regeneration medium (RD)    Sorbitol           186 g/L    Agarose            10 g/L    Glucose            20 g/L    Yeast nitrogen base                       1.34 g/L    Biotin             400 ug/L    Histidine assay medium*                       2 g/L    Glutamic acid      50 mg/L    Methionine         50 mg/L    Lysine             50 mg/L    Leucine            50 mg/L    Isoleucine         50 mg/L    ______________________________________     *DIFCO Ltd     For base plates use agarose at 20 g/L

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    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 178    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 229 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..229    (D) OTHER INFORMATION: /codon.sub.-- start= 1    /product= "LD78 SYNTHTIC GENE"    (ix) FEATURE:    (A) NAME/KEY: 3'UTR    (B) LOCATION: 223..225    (D) OTHER INFORMATION: /function= "NON-TRANSLATED STOP    CODON"    (ix) FEATURE:    (A) NAME/KEY: 3'UTR    (B) LOCATION: 226..228    (D) OTHER INFORMATION: /function= "NON-TRANSLATED STOP    CODON"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    AGCTTGGATAAAAGATCCTTGGCTGCTGACACTCCAACCGCTTGTTGT48    SerLeuAspLysArgSerLeuAlaAlaAspThrProThrAlaCysCys    151015    TTCTCTTACACCTCTAGACAAATTCCACAAAATTTCATTGCTGACTAC96    PheSerTyrThrSerArgGlnIleProGlnAsnPheIleAlaAspTyr    202530    TTTGAAACTTCTTCTCAATGTTCCAAGCCAGGTGTCATCTTCTTGACT144    PheGluThrSerSerGlnCysSerLysProGlyValIlePheLeuThr    354045    AAGCGCTCGAGACAAGTCTGTGCTGACCCATCTGAAGAATGGGTTCAA192    LysArgSerArgGlnValCysAlaAspProSerGluGluTrpValGln    505560    AAATATGTTTCTGACTTGGAATTGTCTGCCTAATAAG229    LysTyrValSerAspLeuGluLeuSerAla    6570    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 69 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    SerLeuAlaAlaAspThrProThrAlaCysCysPheSerTyrThrSer    151015    ArgGlnIleProGlnAsnPheIleAlaAspTyrPheGluThrSerSer    202530    GlnCysSerLysProGlyValIlePheLeuThrLysArgSerArgGln    354045    ValCysAlaAspProSerGluGluTrpValGlnLysTyrValSerAsp    505560    LeuGluLeuSerAla    65    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 229 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (iii) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    CTTATTAGGCAGACAATTCCAAGTCAGAAACATATTTTTGAACCCATTCTTCAGATGGGT60    CAGCACAGACTTGTCTCGAGCGCTTAGTCAAGAAGATGACACCTGGCTTGGAACATTGAG120    AAGAAGTTTCAAAGTAGTCAGCAATGAAATTTTGTGGAATTTGTCTAGAGGTGTAAGAGA180    AACAACAAGCGGTTGGAGTGTCAGCAGCCAAGGATCTTTTATCCAAGCT229    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 45 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..45    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF SYNTHETIC LD78 GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    AGCTTGGATAAAAGATCCTTGGCTGCTGACACTCCAACCGCTTGT45    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 48 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..48    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF SYNTHETIC LD78 GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    AGAAACAACAAGCGGTTGGAGTGTCAGCAGCCAAGGATCTTTTATCCA48    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 50 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..50    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF SYNTHETIC LD78 GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    TGTTTCTCTTACACCTCTAGACAAATTCCACAAAATTTCATTGCTGACTA50    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 50 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..50    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF SYNTHETIC LD78 GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    TTCAAAGTAGTCAGCAATGAAATTTTGTGGAATTTGTCTAGAGGTGTAAG50    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 48 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..48    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF LD78 SYNTHETIC GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    CTTTGAAACTTCTTCTCAATGTTCCAAGCCAGGTGTCATCTTCTTGAC48    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 48 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..48    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF SYNTHETIC LD78 GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    GCGCTTAGTCAAGAAGATGACACCTGGCTTGGAACATTGAGAAGAAGT48    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 46 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..46    (D) OTHER INFORMATION: /product= "OLIGOMER FOR THE    CONSTRUCTION OF LD78 SYNTHETIC GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    TAAGCGCTCGAGACAAGTCTGTGCTGACCCATCTGAAGAATGGGTT46    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 46 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..46    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF SYNTHETIC LD78 GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    ATTTTTGAACCCATTCTTCAGATGGGTCAGCACAGACTTGTCTCGA46    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..40    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF SYNTHETIC LD78 GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    CAAAAATATGTTTCTGACTTGGAATTGTCTGCCTAATAAG40    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 37 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..37    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF SYNTHETIC LD78 GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    GATCCTTATTAGGCAGACAATTCCAAGTCAGAAACAT37    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    GTTTTCCCAGTCACGAC17    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7859 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: circular    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    TTCCCATGTCTCTACTGGTGGTGGTGCTTCTTTGGAATTATTGGAAGGTAAGGAATTGCC60    AGGTGTTGCTTTCTTATCCGAAAAGAAATAAATTGAATTGAATTGAAATCGATAGATCAA120    TTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTG180    AATATTTTTTATTTATATACGTATATATAGACTATTATTTACTTTTAATAGATTATTAAG240    ATTTTTATTAAAAAAAAATTCGTCCCTCTTTTTAATGCCTTTTATGCAGTTTTTTTTTCC300    CATTCGATATTTCTATGTTCGGGTTTCAGCGTATTTTAAGTTTAATAACTCGAAAATTCT360    GCGTTTCGAAAAAGCTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCG420    TATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCG480    GCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAA540    CGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGC600    GTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTC660    AAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAG720    CTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCT780    CCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTA840    GGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGC900    CTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGC960    AGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTT1020    GAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCT1080    GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGC1140    TGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCA1200    AGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTA1260    AGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAA1320    ATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATG1380    CTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTG1440    ACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGC1500    AATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGC1560    CGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAA1620    TTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGC1680    CATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGG1740    TTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTC1800    CTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTAT1860    GGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGG1920    TGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCC1980    GGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGG2040    AAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGAT2100    GTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGG2160    GTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATG2220    TTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCT2280    CATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCAC2340    ATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTA2400    TAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCTGAACCAGTCCTAAAAC2460    GAGTAAATAGGACCGGCAATTCTTCAAGCAATAAACAGGAATACCAATTATTAAAAGATA2520    ACTTAGTCAGATCGTACAATAAAGCTAGCTTTGAAGAAAAATGCGCCTTATTCAATCTTT2580    GCTATAAAAAATGGCCCAAAATCTCACATTGGAAGACATTTGATGACCTCATTTCTTTCA2640    ATGAAGGGCCTAACGGAGTTGACTAATGTTGTGGGAAATTGGAGCGATAAGCGTGCTTCT2700    GCCGTGGCCAGGACAACGTATACTCATCAGATAACAGCAATACCTGATCACTACTTCGCA2760    CTAGTTTCTCGGTACTATGCATATGATCCAATATCAAAGGAAATGATAGCATTGAAGGAT2820    GAGACTAATCCAATTGAGGAGTGGCAGCATATAGAACAGCTAAAGGGTAGTGCTGAAGGA2880    AGCATACGATACCCCGCATGGAATGGGATAATATCACAGGAGGTACTAGACTACCTTTCA2940    TCCTACATAAATAGACGCATATAAGTACGCATTTAAGCATAAACACGCACTATGCCGTTC3000    TTCTCATGTATATATATATACAGGCAACACGCAGATATAGGTGCGACGTGAACAGTGAGC3060    TGTATGTGCGCAGCTCGCGTTGCATTTTCGGAAGCGCTCGTTTTCGGAAACGCTTTGAAG3120    TTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAGAGCGCTTTTGAAAA3180    CCAAAAGCGCTCTGAAGACGCACTTTCAAAAAACCAAAAACGCACCGGACTGTAACGAGC3240    TACTAAAATATTGCGAATACCGCTTCCACAAACATTGCTCAAAAGTATCTCTTTGCTATA3300    TATCTCTGTGCTATATCCCTATATAACCTACCCATCCACCTTTCGCTCCTTGAACTTGCA3360    TCTAAACTCGACCTCTACATTTTTTATGTTTATCTCTAGTATTACTCTTTAGACAAAAAA3420    ATTGTAGTAAGAACTATTCATAGAGTGAATCGAAAACAATACGAAAATGTAAACATTTCC3480    TATACGTAGTATATAGAGACAAAATAGAAGAAACCGTTCATAATTTTCTGACCAATGAAG3540    AATCATCAACGCTATCACTTTCTGTTCACAAAGTATGCGCAATCCACATCGGTATAGAAT3600    ATAATCGGGGATGCCTTTATCTTGAAAAAATGCACCCGCAGCTTCGCTAGTAATCAGTAA3660    ACGCGGGAAGTGGAGTCAGGCTTTTTTTATGGAAGAGAAAATAGACACCAAAGTAGCCTT3720    CTTCTAACCTTAACGGACCTACAGTGCAAAAAGTTATCAAGAGACTGCATTATAGAGCGC3780    ACAAAGGAGAAAAAAAGTAATCTAAGATGCTTTGTTAGAAAAATAGCGCTCTCGGGATGC3840    ATTTTTGTAGAACAAAAAAGAAGTATAGATTCTTTGTTGGTAAAATAGCGCTCTCGCGTT3900    GCATTTCTGTTCTGTAAAAATGCAGCTCAGATTCTTTGTTTGAAAAATTAGCGCTCTCGC3960    GTTGCATTTTTGTTTTACAAAAATGAAGCACAGATTCTTCGTTGGTAAAATAGCGCTTTC4020    GCGTTGCATTTCTGTTCTGTAAAAATGCAGCTCAGATTCTTTGTTTGAAAAATTAGCGCT4080    CTCGCGTTGCATTTTTGTTCTACAAAATGAAGCACAGATGCTTCGTTAACAAAGATATGC4140    TATTGAAGTGCAAGATGGAAACGCAGAAAATGAACCGGGGATGCGACGTGCAAGATTACC4200    TATGCAATAGATGCAATAGTTTCTCCAGGAACCGAAATACATACATTGTCTTCCGTAAAG4260    CGCTAGACTATATATTATTATACAGGTTCAAATATACTATCTGTTTCAGGGAAAACTCCC4320    AGGTTCGGATGTTCAAAATTCAATGATGGGTAACAAGTACGATCGTAAATCTGTAAAACA4380    GTTTGTCGGATATTAGGCTGTATCTCCTCAAAGCGTATTCGAATATCATTGAGAAGCTGC4440    ATTTTTTTTTTTTTTTATATATATTTCAAGGATATACCATTGTAATGCCTGCCCCTAAGA4500    AGATCGTCGTTTTGCCAGGTGACCACGTTGGTCAAGAAATCACAGCCGAAGCCATTAAGG4560    TTCTTAAAGCTATTTCTGATGTTCGTTCCAATGTCAAGTTCGATTTCGAAAATCATTTAA4620    TTGGTGGTGCTGCTATCGATGCTACAGGTGTTCCACTTCCAGATGAGGCGCTGGAAGCCT4680    CCAAGAAGGCTGATGCCGTTTTGTTAGGTGCTGTGGGTGGTCCTAAATGGGGTACCGGTA4740    GTGTTAGACCTGAACAAGGTTTACTAAAAATCCGTAAAGAACTTCAATTGTACGCCAACT4800    TAAGACCATGTAACTTTGCATCCGACTCTCTTTTAGACTTATCTCCAATCAAGCCACAAT4860    TTGCTAAAGGTACTGACTTCGTTGTTGTTAGAGAATTAGTGGGAGGTATTTACTTTGGTA4920    AGAGAAAGGAAGACGATGGTGATGGTGTCGCTTGGGATAGTGAACAATACACCGTTCCAG4980    AAGTGCAAAGAATCACAAGAATGGCCGCTTTCATGGCCCTACAACATGAGCCACCATTGC5040    CTATTTGGTCCTTGGATAAAGCTAATGTTTTGGCCTCTTCAAGATTATGGAGAAAAACTG5100    TGGAGGAAACCATCAAGAACGAATTCCCTACATTGAAAGTTCAACATCAATTGATTGATT5160    CTGCCGCCATGATCCTAGTTAAGAACCCAACCCACCTAAATGGTATTATAATCACCAGCA5220    ACATGTTTGGTGATATCATCTCCGATGAAGCCTCCGTTATCCCAGGCTCCTTGGGTTTGT5280    TGCCATCTGCGTCCTTGGCCTCTTTGCCAGACAAGAACACCGCATTTGGTTTGTACGAAC5340    CATGCCATGGTTCCGCTCCAGATTTGCCAAAGAATAAGGTCAACCCTATCGCCACTATCT5400    TGTCTGCTGCAATGATGTTGAAATTGTCATTGAACTTGCCTGAAGAAGGTAAAGCCATTG5460    AAGATGCAGTTAAAAAGGTTTTGGATGCAGGTATCAGAACTGGTGATTTAGGTGGTTCCA5520    ACAGTACCACCGAAGTCGGTGATGCTGTCGCCGAAGAAGTTAAGAAAATCCTTGCTTAAA5580    AAGATTCTCTTTTTTTATGATATTTGTACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA5640    AAAAAAAAAAAAAAAAAAAAAAAATGCAGCGTCACATCGGATAATAATGATGGCAGCCAT5700    TGTAGAAGTGCCTTTTGCATTTCTAGTCTCTTTCTCGGTCTAGCTAGTTTTACTACATCG5760    CGAAGATAGAATCTTAGATCACACTGCCTTTGCTGAGCTGGATCAATAGAGTAACAAAAG5820    AGTGGTAAGGCCTCGTTAAAGGACAAGGACCTGAGCGGAAGTGTATCGTACAGTAGACGG5880    AGTATACTAGTATAGTCTATAGTCCGTGGAATTCTCATGTTTGACAGCTTATCATCGATA5940    AGCTAGCTTTCTAACTGATCTATCCAAAACTGAAAATTACATTCTTGATTAGGTTTATCA6000    CAGGCAAATGTAATTTGTGGTATTTTGCCGTTCAAAATCTGTAGAATTTTCTCATTGGTC6060    ACATTACAACCTGAAAATACTTTATCTACAATCATACCATTCTTAATAACATGTCCCCTT6120    AATACTAGGATCAGGCATGAACGCATCACAGACAAAATCTTCTTGACAAACGTCACAATT6180    GATCCCTCCCCATCCGTTATCACAATGACAGGTGTCATTTTGTGCTCTTATGGGACGATC6240    CTTATTACCGCTTTCATCCGGTGATTGACCGCCACAGAGGGGCAGAGAGCAATCATCACC6300    TGCAAACCCTTCTATACACTCACATCTACCAGTGATCGAATTGCATTCAGAAAACTGTTT6360    GCATTCAAAAATAGGTAGCATACAATTAAAACATGGCGGGCATGTATCATTGCCCTTATC6420    TTGTGCAGTTAGACGCGAATTTTTCGAAGAAGTACCTTCAAAGAATGGGGTCTTATCTTG6480    TTTTGCAAGTACCACTGAGCAGGATAATAATAGAAATGATAATATACTATAGTAGAGATA6540    ACGTCGATGACTTCCCATACTGTAATTGCTTTTAGTTGTGTATTTTTAGTGTGCAAGTTT6600    CTGTAAATCGATTAATTTTTTTTTCTTTCCTCTTTTTATTAACCTTAATTTTTATTTTAG6660    ATTCCTGACTTCAACTCAAGACGCACAGATATTATAACATCTGCATAATAGGCATTTGCA6720    AGAATTACTCGTGAGTAAGGAAAGAGTGAGGAACTATCGCATACCTGCATTTAAAGATGC6780    CGATTTGGGCGCGAATCCTTTATTTTGGCTTCACCCTCATACTATTATCAGGGCCAGAAA6840    AAGGAAGTGTTTCCCTCCTTCTTGAATTGATGTTACCCTCATAAAGCACGTGGCCTCTTA6900    TCGAGAAAGAAATTACCGTCGCTCGTGATTTGTTTGCAAAAAGAACAAAACTGAAAAAAC6960    CCAGACACGCTCGACTTCCTGTCTTCCTATTGATTGCAGCTTCCAATTTCGTCACACAAC7020    AAGGTCCTAGCGACGGCTCACAGGTTTTGTAACAAGCAATCGAAGGTTCTGGAATGGCGG7080    GGAAAGGGTTTAGTACCACATGCTATGATGCCCACTGTGATCTCCAGAGCAAAGTTCGTT7140    CGATCGTACTGTACTCTCTCTCTTTCAAACAGAATTGTCCGAATCGTGTGACAACAACAG7200    CCTGTTCTCACACACTCTTTTCTTCTAACCAAGGGGGTGGTTTAGTTTAGTAGAACCTCG7260    TGAAACTTACATTTACATATATATAAACTTGCATAAATTGGTCAATGGAAGAAATACATA7320    TTTGGTCTTTTCTAATTCGTAGTTTTTCAAGTTCTTAGATGCTTTCTTTTTCTCTTTTTT7380    ACAGATCATCAAGGAAGTAATTATCTACTTTTTACAACAAATACAAAAGATCTATGAGAT7440    TTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCA7500    ACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTTAG7560    ATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGT7620    TATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTAAGCTTGG7680    ATAAAAGAAACAGCGACTCTGAATGCCCGCTGAGCCATGATGGCTACTGCCTGCACGACG7740    GTGTATGCATGTATATCGAAGCTCTGGACAAATACGCATGCAACTGCGTAGTTGGTTACA7800    TCGGCGAACGTTGCCAGTACCGCGACCTGAAATGGTGGGAGCTCCGTTAATAAGGATCC7859    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    AGGATGGGGAAAGAGAA17    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 234 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..234    (D) OTHER INFORMATION: /codon.sub.-- start= 1    /product= "MIP-1-ALPHA GENE"    (ix) FEATURE:    (A) NAME/KEY: 3'UTR    (B) LOCATION: 223..225    (D) OTHER INFORMATION: /function= "UNTRANSLATED STOP    CODON"    (ix) FEATURE:    (A) NAME/KEY: 3'UTR    (B) LOCATION: 226..228    (D) OTHER INFORMATION: /function= "NON-TRANSLATED STOP    CODON"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    AGCTTACCTGCCATGGCGCCTTATGGAGCTGACACCCCGACTGCATGC48    SerLeuProAlaMetAlaProTyrGlyAlaAspThrProThrAlaCys    151015    TGCTTCTCCTACAGCCGGAAGATTCCACGCCAATTCATCGTCGACTAT96    CysPheSerTyrSerArgLysIleProArgGlnPheIleValAspTyr    202530    TTTGAAACTAGTAGCCTTTGCTCCCAGCCAGGTGTCATTTTCCTGACT144    PheGluThrSerSerLeuCysSerGlnProGlyValIlePheLeuThr    354045    AAGAGAAACCGGCAGATCTGCGCTGACTCCAAAGAGACCTGGGTCCAA192    LysArgAsnArgGlnIleCysAlaAspSerLysGluThrTrpValGln    505560    GAATACATCACTGACCTCGAGCTGAATGCCTGATAGGATCCG234    GluTyrIleThrAspLeuGluLeuAsnAlaAspPro    657075    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 74 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    SerLeuProAlaMetAlaProTyrGlyAlaAspThrProThrAla    151015    CysCysPheSerTyrSerArgLysIleProArgGlnPheIleVal    202530    AspTyrPheGluThrSerSerLeuCysSerGlnProGlyValIle    354045    PheLeuThrLysArgAsnArgGlnIleCysAlaAspSerLysGlu    505560    ThrTrpValGlnGluTyrIleThrAspLeuGluLeuAsnAla    6570    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 234 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (iii) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    CGGATCCTATCAGGCATTCAGCTCGAGGTCAGTGATGTATTCTTGGACCCAGGTCTCTTT60    GGAGTCAGCGCAGATCTGCCGGTTTCTCTTAGTCAGGAAAATGACACCTGGCTGGGAGCA120    AAGGCTACTAGTTTCAAAATAGTCGACGATGAATTGGCGTGGAATCTTCCGGCTGTAGGA180    GAAGCAGCATGCAGTCGGGGTGTCAGCTCCATAAGGCGCCATGGCAGGTAAGCT234    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 38 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..38    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF MIP-ALPHA GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    AGCTTACCTGCCATGGCGCCTTATGGAGCTGACACCCC38    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 41 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..41    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCITON OF MIP1-ALPHA GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    TGCAGTCGGGGTGTCAGCTCCATAAGGCGCCATGGCAGGTA41    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 44 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..44    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF MIP1-ALPHA GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    GACTGCATGCTGCTTCTCCTACAGCCGGAAGATTCCACGCCAAT44    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 44 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..43    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF MIP1-ALPHA GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    ACGATGAATTGGCGTGGAATCTTCCGGCTGTAGGAGAAGCAGCA44    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..39    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF MIP1-ALPHA GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    TCATCGTCGACTATTTTGAAACTAGTAGCCTTTGCTCCC39    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..39    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF MIP1-ALPHA GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    CCTGGCTGGGAGCAAAGGCTACTAGTTTCAAAATAGTCG39    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 37 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..37    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF MIP1-ALPHA GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    AGCCAGGTGTCATTTTCCTGACTAAGAGAAACCGGCA37    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 37 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..37    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF MIP1-ALPHA GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    GCAGATCTGCCGGTTTCTCTTAGTCAGGAAAATGACA37    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 44 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..44    (D) OTHER INFORMATION: /product= "OLIGOMER FOR THE    CONSTRUCTION OF MIP1-ALPHA GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    GATCTGCGCTGACTCCAAAGAGACCTGGGTCCAAGAATACATCA44    (2) INFORMATION FOR SEQ ID NO:29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 44 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..44    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF MIP1-ALPHA GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    AGGTCAGTGATGTATTCTTGGACCCAGGTCTCTTTGGAGTCAGC44    (2) INFORMATION FOR SEQ ID NO:30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 32 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..32    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF MIP1-ALPHA SYNTHETIC GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    CTGACCTCGAGCTGAATGCCTGATAGGATCCG32    (2) INFORMATION FOR SEQ ID NO:31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 29 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..29    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF MIP1-ALPHA GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    AATTCGGATCCTATCAGGCATTCAGCTCG29    (2) INFORMATION FOR SEQ ID NO:32:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..15    (D) OTHER INFORMATION: /product= "TOP STRAND OF    OLIGONUCLEOTIDE ADAPTOR"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    AGCTTGGATAAAAGA15    (2) INFORMATION FOR SEQ ID NO:33:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..11    (D) OTHER INFORMATION: /product= "BOTTOM STRAND OF    OLIGONUCLEOTIDE ADAPTOR"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    TCTTTTATCCA11    (2) INFORMATION FOR SEQ ID NO:34:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 229 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..228    (D) OTHER INFORMATION: /codon.sub.-- start= 1    /product= "HUMAN ACT-2 SYNTHETIC GENE"    (ix) FEATURE:    (A) NAME/KEY: 3'UTR    (B) LOCATION: 223..225    (D) OTHER INFORMATION: /function= "NON-TRANSLATED STOP    CODON"    (ix) FEATURE:    (A) NAME/KEY: 3'UTR    (B) LOCATION: 226..228    (D) OTHER INFORMATION: /function= "NON-TRANSLATED STOP    CODON"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    AGCTTGGATAAAAGAGCACCAATGGGTTCAGACCCTCCAACCGCATGC48    SerLeuAspLysArgAlaProMetGlySerAspProProThrAlaCys    151015    TGCTTTTCTTACACCGCTAGGAAGTTGCCTAGAAACTTTGTGGTCGAC96    CysPheSerTyrThrAlaArgLysLeuProArgAsnPheValValAsp    202530    TACTATGAGACCTCTTCTTTGTGCTCCCAGCCAGCTGTGGTATTCCAA144    TyrTyrGluThrSerSerLeuCysSerGlnProAlaValValPheGln    354045    ACCAAAAGATCCAAGCAAGTCTGTGCTGACCCGAGTGAATCCTGGGTC192    ThrLysArgSerLysGlnValCysAlaAspProSerGluSerTrpVal    505560    CAGGAGTACGTGTATGACTTGGAATTGAACTGATAAG229    GlnGluTyrValTyrAspLeuGluLeuAsn    6570    (2) INFORMATION FOR SEQ ID NO:35:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 74 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    SerLeuAspLysArgAlaProMetGlySerAspProProThrAlaCys    151015    CysPheSerTyrThrAlaArgLysLeuProArgAsnPheValValAsp    202530    TyrTyrGluThrSerSerLeuCysSerGlnProAlaValValPheGln    354045    ThrLysArgSerLysGlnValCysAlaAspProSerGluSerTrpVal    505560    GlnGluTyrValTyrAspLeuGluLeuAsn    6570    (2) INFORMATION FOR SEQ ID NO:36:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 229 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    CTTATCAGTTCAATTCCAAGTCATACACGTACTCCTGGACCCAGGATTCACTCGGGTCAG60    CACAGACTTGCTTGGATCTTTTGGTTTGGAATACCACAGCTGGCTGGGAGCACAAAGAAG120    AGGTCTCATAGTAGTCGACCACAAAGTTTCTAGGCAACTTCCTAGCGGTGTAAGAAAAGC180    AGCATGCGGTTGGAGGGTCTGAACCCATTGGTGCTCTTTTATCCAAGCT229    (2) INFORMATION FOR SEQ ID NO:37:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 46 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..46    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF HUMAN ACT-2 GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    AGCTTGGATAAAAGAGCACCAATGGGTTCAGACCCTCCAACCGCAT46    (2) INFORMATION FOR SEQ ID NO:38:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 45 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..45    (D) OTHER INFORMATION: /product= "OLIGOMER FOR    CONSTRUCTION OF HUMAN ACT-2 GENE"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    AGCATGCGGTTGGAGGGTCTGAACCCATTGGTGCTCTTTTATCCA45    (2) INFORMATION FOR SEQ ID NO:39:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 47 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..47    (D) OTHER INFORMATION: /product= "Oligomer for    construction of human ACT-2 gene"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    GCTGCTTTTCTTACACCGCTAGGAAGTTGCCTAGAAACTTTGTGGTC47    (2) INFORMATION FOR SEQ ID NO:40:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 51 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..51    (D) OTHER INFORMATION: /product= "Oligomer for    construction of human ACT-2 gene"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    AGTAGTCGACCACAAAGTTTCTAGGCAACTTCCTAGCGGTGTAAGAAAAGC51    (2) INFORMATION FOR SEQ ID NO:41:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 46 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..46    (D) OTHER INFORMATION: /product= "Oligomer for    construction of human ACT-2 gene"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    GACTACTATGAGACCTCTTCTTTGTGCTCCCAGCCAGCTGTGGTAT46    (2) INFORMATION FOR SEQ ID NO:42:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 46 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..46    (D) OTHER INFORMATION: /product= "Oligomer for    construction of human ACT-2 gene"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:    GTTTGGAATACCACAGCTGGCTGGGAGCACAAAGAAGAGGTCTCAT46    (2) INFORMATION FOR SEQ ID NO:43:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 47 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..47    (D) OTHER INFORMATION: /product= "Oligomer for    construction of human ACT-2 gene"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:    TCCAAACCAAAAGATCCAAGCAAGTCTGTGCTGACCCGAGTGAATCC47    (2) INFORMATION FOR SEQ ID NO:44:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 47 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..47    (D) OTHER INFORMATION: /product= "Oligomer for    construction of human ACT-2 gene"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:    GGACCCAGGATTCACTCGGGTCAGCACAGACTTGCTTGGATCTTTTG47    (2) INFORMATION FOR SEQ ID NO:45:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 43 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..43    (D) OTHER INFORMATION: /product= "Oligomer for    construction of human ACT-2 gene"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:    TGGGTCCAGGAGTACGTGTATGACTTGGAATTGAACTGATAAG43    (2) INFORMATION FOR SEQ ID NO:46:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..40    (D) OTHER INFORMATION: /product= "Oligomer for    construction of human ACT-2 gene"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:    GATCCTTATCAGTTCAATTCCAAGTCATACACGTACTCCT40    (2) INFORMATION FOR SEQ ID NO:47:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:    GTTTTCCCAGTCACGAC17    (2) INFORMATION FOR SEQ ID NO:48:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:    GCACAGACTTCTCTCGAGCGCT22    (2) INFORMATION FOR SEQ ID NO:49:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..30    (D) OTHER INFORMATION: /product= "BB6299 oligomer    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:    GACTTGTCTCGATTGCTCAGTCAAGAAGAT30    (2) INFORMATION FOR SEQ ID NO:50:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..22    (D) OTHER INFORMATION: /product= "BB6300 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:    AAACAACAAGAGGTTGGAGTGT22    (2) INFORMATION FOR SEQ ID NO:51:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..25    (D) OTHER INFORMATION: /product= "BB6381 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:    GAAGAAGTTTCABAGTAGTCAGCAA25    (2) INFORMATION FOR SEQ ID NO:52:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..25    (D) OTHER INFORMATION: /product= "BB6302 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:    GTGGAATTTGAGAAGAGGTGTAAGA25    (2) INFORMATION FOR SEQ ID NO:53:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..27    (D) OTHER INFORMATION: /product= "BB6303 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:    GTAGTCAGCAGTGTTATTTTGTGGAAT27    (2) INFORMATION FOR SEQ ID NO:54:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..25    (D) OTHER INFORMATION: /product= "BB6625 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:    TTTCAAAGTAGRCAGCAATGAAATT25    (2) INFORMATION FOR SEQ ID NO:55:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..24    (D) OTHER INFORMATION: /product= "BB6301 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:    AGGTGTAAGATTGACAACAAGCGG24    (2) INFORMATION FOR SEQ ID NO:56:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..25    (D) OTHER INFORMATION: /product= "BB6382 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:    AGTAGTCAGCABTGAAATTTTGTGG25    (2) INFORMATION FOR SEQ ID NO:57:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..24    (D) OTHER INFORMATION: /product= "BB6383 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:    TAGTCAAGAATCTGACACCTGGCT24    (2) INFORMATION FOR SEQ ID NO:58:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..26    (D) OTHER INFORMATION: /product= "BB6384 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:    GCACAGACTTGTTCCGAGCGCTTAGT26    (2) INFORMATION FOR SEQ ID NO:59:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 35 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..35    (D) OTHER INFORMATION: /product= "BB6385 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:    AATTCCAAGTTAGAAACATATTGTTGAACCCATTC35    (2) INFORMATION FOR SEQ ID NO:60:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..25    (D) OTHER INFORMATION: /product= "BB6345 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:    GAAGAAGTTTCTTCGTAGTCAGCAA25    (2) INFORMATION FOR SEQ ID NO:61:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..27    (D) OTHER INFORMATION: /product= "BB7015 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:    TGAGAAGAAGTTTCTTCGTAGTCAGCA27    (2) INFORMATION FOR SEQ ID NO:62:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..27    (D) OTHER INFORMATION: /product= "BB9112 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:    TTGAACCCAGCGGCGAGATGGGTCAGC27    (2) INFORMATION FOR SEQ ID NO:63:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..24    (D) OTHER INFORMATION: /product= "BB9109 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:    TTGAGAAGAAGTTCTAAAGTAGTC24    (2) INFORMATION FOR SEQ ID NO:64:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..24    (D) OTHER INFORMATION: /product= "BB9110 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:    ATTTTGTGGAATTTCTCTAGAGGT24    (2) INFORMATION FOR SEQ ID NO:65:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..30    (D) OTHER INFORMATION: /product= "BB9111 Oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:    ATTTTGTGGAATTTCAGAAGAGGTGTAAGA30    (2) INFORMATION FOR SEQ ID NO:66:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..30    (D) OTHER INFORMATION: /product= "BB9104 Oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:    AGCAGCCAAGGAAGCAGATCTTTTATCCAA30    (2) INFORMATION FOR SEQ ID NO:67:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..36    (D) OTHER INFORMATION: /product= "BB9105 Oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:    GTCAGCAGCCAATGGAGCAGACAATCTTTTATCCAA36    (2) INFORMATION FOR SEQ ID NO:68:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..24    (D) OTHER INFORMATION: /product= "BB9106 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:    TGGAGTGTCAGCTCTTTTATCCAA24    (2) INFORMATION FOR SEQ ID NO:69:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..30    (D) OTHER INFORMATION: /product= "BB9103 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:    GTCAGCAGCCAATGGAGCTCTTTTATCCAA30    (2) INFORMATION FOR SEQ ID NO:70:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 48 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..48    (D) OTHER INFORMATION: /product= "BB9108 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:    ACAGACTTGTCTACCGCGCTTAGTCAAGAAGATGACAGATGGCTTGGA48    (2) INFORMATION FOR SEQ ID NO:71:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..24    (D) OTHER INFORMATION: /product= "BB9107 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:    AATTTGTCTAGAGAAGTAAGAGAA24    (2) INFORMATION FOR SEQ ID NO:72:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..21    (D) OTHER INFORMATION: /product= "BB9512 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:    CAGCACAGACAGATCTCGAGC21    (2) INFORMATION FOR SEQ ID NO:73:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..18    (D) OTHER INFORMATION: /product= "BB9432 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:    CAAAGTAGGAAGCAATGA18    (2) INFORMATION FOR SEQ ID NO:74:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB9519 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:    GTGTAAGAGGCACAACAAG19    (2) INFORMATION FOR SEQ ID NO:75:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB9527 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:    GAAGTTTCAGCGTAGTCAG19    (2) INFORMATION FOR SEQ ID NO:76:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..21    (D) OTHER INFORMATION: /product= "BB9431 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:    GTAGTCAGCAGCGAAATTTTG21    (2) INFORMATION FOR SEQ ID NO:77:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB9534 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:    GTCAAGAAGGCGACACCTG19    (2) INFORMATION FOR SEQ ID NO:78:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..21    (D) OTHER INFORMATION: /product= "BB9437 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:    CACAGACTTGAGACGAGCGCT21    (2) INFORMATION FOR SEQ ID NO:79:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..22    (D) OTHER INFORMATION: /product= "BB9433 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:    GAGAAGAAGTAGAAAAGTAGTC22    (2) INFORMATION FOR SEQ ID NO:80:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..21    (D) OTHER INFORMATION: /product= "BB9506 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:    TTTGTGGAATAGATCTAGAGG21    (2) INFORMATION FOR SEQ ID NO:81:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..23    (D) OTHER INFORMATION: /product= "BB10194 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:    GGTTGGAGTGCGAGCAGCCAAGG23    (2) INFORMATION FOR SEQ ID NO:82:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..22    (D) OTHER INFORMATION: /product= "BB10195 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:    GGAATTTGTTCAGAGGTGTAAG22    (2) INFORMATION FOR SEQ ID NO:83:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..27    (D) OTHER INFORMATION: /product= "BB10196 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:    GCACAGACTTGTCTTTCGCGCTTAGTC27    (2) INFORMATION FOR SEQ ID NO:84:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 29 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..29    (D) OTHER INFORMATION: /product= "BB10197 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:    GGAGTGTCAGCAGCTTCGGATCTTTTATC29    (2) INFORMATION FOR SEQ ID NO:85:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..22    (D) OTHER INFORMATION: /product= "BB10198 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:    GGAGTGTCAGCTTCCAAGGATC22    (2) INFORMATION FOR SEQ ID NO:86:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..23    (D) OTHER INFORMATION: /product= "BB10199 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:    GGTTGGAGTGTCTTCAGCCAAGG23    (2) INFORMATION FOR SEQ ID NO:87:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..22    (D) OTHER INFORMATION: /product= "BB10200 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:    GGAATTTCTTCAGAGGTGTAAG22    (2) INFORMATION FOR SEQ ID NO:88:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..28    (D) OTHER INFORMATION: /product= "BB10201 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:    CCTTATTAGGCAGATTCTTCCAAGTCAG28    (2) INFORMATION FOR SEQ ID NO:89:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..20    (D) OTHER INFORMATION: /product= "BB9537 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:    GACTTGTCTAGCGCGCTTAG20    (2) INFORMATION FOR SEQ ID NO:90:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB9497 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:    GTCAGCAGCAGCGGATCTT19    (2) INFORMATION FOR SEQ ID NO:91:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..17    (D) OTHER INFORMATION: /product= "BB9498 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:    GTCAGCAGACAAGGATC17    (2) INFORMATION FOR SEQ ID NO:92:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..18    (D) OTHER INFORMATION: /product= "BB9499 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:    GAGTGTCAGAAGCCAAGG18    (2) INFORMATION FOR SEQ ID NO:93:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..22    (D) OTHER INFORMATION: /product= "BB9517 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:    ATTAGGCAGAGGCTTCCAAGTC22    (2) INFORMATION FOR SEQ ID NO:94:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 34 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..34    (D) OTHER INFORMATION: /product= "BB9781 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:    GAGAAACAACAAGCGGTAGATCTTTTATCCAAGC34    (2) INFORMATION FOR SEQ ID NO:95:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..20    (D) OTHER INFORMATION: /product= "BB9430 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:    GTTGGAGTGGAAGCAGCCAA20    (2) INFORMATION FOR SEQ ID NO:96:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..18    (D) OTHER INFORMATION: /product= "BB9525 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:    CAGCAATGGCATTTTGTG18    (2) INFORMATION FOR SEQ ID NO:97:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..21    (D) OTHER INFORMATION: /product= "BB9435 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:    GTCTCGAGCGAGAAGTCAAGA21    (2) INFORMATION FOR SEQ ID NO:98:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..18    (D) OTHER INFORMATION: /product= "BB9436 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:    GTCTCGAGGACTTAGTCA18    (2) INFORMATION FOR SEQ ID NO:99:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..22    (D) OTHER INFORMATION: /product= "BB9423 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:    GAACCCATTCAGAAGATGGGTC22    (2) INFORMATION FOR SEQ ID NO:100:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..21    (D) OTHER INFORMATION: /product= "BB9424 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:    TTTGAACCCAAGATTCAGATG21    (2) INFORMATION FOR SEQ ID NO:101:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..21    (D) OTHER INFORMATION: /product= "BB9425 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:    CAGAAACATAAGATTGAACCC21    (2) INFORMATION FOR SEQ ID NO:102:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..20    (D) OTHER INFORMATION: /product= "BB9427 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:    CAATTCCAAGGAAGAAACAT20    (2) INFORMATION FOR SEQ ID NO:103:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..17    (D) OTHER INFORMATION: /product= "BB9503 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:    CCTTATTAGTCAGAAAC17    (2) INFORMATION FOR SEQ ID NO:104:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..33    (D) OTHER INFORMATION: /product= "BB9443 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:    TTGAGAAGAAGTTCTAAAGTAGGCAGCAATGAA33    (2) INFORMATION FOR SEQ ID NO:105:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..20    (D) OTHER INFORMATION: /product= "BB9434 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:    GACACCTGGAGAGGAACATT20    (2) INFORMATION FOR SEQ ID NO:106:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..22    (D) OTHER INFORMATION: /product= "BB9228 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:    CAGACAATTCAGCGTCAGAAAC22    (2) INFORMATION FOR SEQ ID NO:107:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..20    (D) OTHER INFORMATION: /product= "BB9429 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:    GGCAGACAAAGACAAGTCAG20    (2) INFORMATION FOR SEQ ID NO:108:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB9495 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:    CTTATTAGGAAGACAATTC19    (2) INFORMATION FOR SEQ ID NO:109:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB9496 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:    CAGCCAAGGCTCTTTTATC19    (2) INFORMATION FOR SEQ ID NO:110:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..20    (D) OTHER INFORMATION: /product= "BB9509 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:    CTTGGAACAAGAAGAAGAAG20    (2) INFORMATION FOR SEQ ID NO:111:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:    GTCAGAAACAGCTTTTTGA19    (2) INFORMATION FOR SEQ ID NO:112:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB9529 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:    CATTGAGAAGCAGTTTCAA19    (2) INFORMATION FOR SEQ ID NO:113:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB9530 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:    GAACATTGAGCAGAAGTTT19    (2) INFORMATION FOR SEQ ID NO:114:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..21    (D) OTHER INFORMATION: /product= "BB9536 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:    GCGCTTAGTAGCGAAGATGAC21    (2) INFORMATION FOR SEQ ID NO:115:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..22    (D) OTHER INFORMATION: /product= "BB9422 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:    CTTCAGATGGAGAAGCACAGAC22    (2) INFORMATION FOR SEQ ID NO:116:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..21    (D) OTHER INFORMATION: /product= "BB9426 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:    CAAGTCAGAAGCATATTTTTG21    (2) INFORMATION FOR SEQ ID NO:117:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..17    (D) OTHER INFORMATION: /product= "BB9504 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:    GGTGTAAGCGAAACAAC17    (2) INFORMATION FOR SEQ ID NO:118:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB9505 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:    ATTTGTCTAGCGGTGTAAG19    (2) INFORMATION FOR SEQ ID NO:119:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..20    (D) OTHER INFORMATION: /product= "BB9507 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:    GAAATTTTGAGCAATTTGTC20    (2) INFORMATION FOR SEQ ID NO:120:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..18    (D) OTHER INFORMATION: /product= "BB9510 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:    CTGGCTTGGCACATTGAG18    (2) INFORMATION FOR SEQ ID NO:121:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..23    (D) OTHER INFORMATION: /product= "BB9514 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:    GAAACATATTTAGAAACCCATTC23    (2) INFORMATION FOR SEQ ID NO:122:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:    ATTAGGCAGCCAATTCCAA19    (2) INFORMATION FOR SEQ ID NO:123:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB9520 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:    CTAGAGGTGGCAGAGAAAC19    (2) INFORMATION FOR SEQ ID NO:124:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB9522 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:    TTTTGTGGAGCTTGTCTAG19    (2) INFORMATION FOR SEQ ID NO:125:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..20    (D) OTHER INFORMATION: /product= "BB9531 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:    GATGACACCAGCCTTGGAAC20    (2) INFORMATION FOR SEQ ID NO:126:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..20    (D) OTHER INFORMATION: /product= "BB9532 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:    GAAGATGACAGCTGGCTTGG20    (2) INFORMATION FOR SEQ ID NO:127:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..18    (D) OTHER INFORMATION: /product= "BB9533 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:    AGAAGATGGCACCTGGCT18    (2) INFORMATION FOR SEQ ID NO:128:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..17    (D) OTHER INFORMATION: /product= "BB9500 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:    GGTTGGAGCGTCAGCAG17    (2) INFORMATION FOR SEQ ID NO:129:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..22    (D) OTHER INFORMATION: /product= "BB9523 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129:    CAATGAAATTAGATGGAATTTG22    (2) INFORMATION FOR SEQ ID NO:130:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..17    (D) OTHER INFORMATION: /product= "BB9511 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:    GCGCTTAGCCAAGAAGA17    (2) INFORMATION FOR SEQ ID NO:131:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB9501 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131:    CAAGCGGTAGCAGTGTCAG19    (2) INFORMATION FOR SEQ ID NO:132:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..18    (D) OTHER INFORMATION: /product= "BB9502 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:    ACAAGCGGCTGGAGTGTC18    (2) INFORMATION FOR SEQ ID NO:133:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..18    (D) OTHER INFORMATION: /product= "BB9508 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133:    GTTTCAAAGGCGTCAGCA18    (2) INFORMATION FOR SEQ ID NO:134:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..20    (D) OTHER INFORMATION: /product= "BB9513 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:    TTCTTCAGATGCGTCAGCAC20    (2) INFORMATION FOR SEQ ID NO:135:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..18    (D) OTHER INFORMATION: /product= "BB9516 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:    CAAGTCAGCAACATATTT18    (2) INFORMATION FOR SEQ ID NO:136:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..17    (D) OTHER INFORMATION: /product= "BB9521 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:    GTCTAGAGGCGTAAGAG17    (2) INFORMATION FOR SEQ ID NO:137:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..18    (D) OTHER INFORMATION: /product= "BB9524 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:    CAATGAAAGATTGTGGAA18    (2) INFORMATION FOR SEQ ID NO:138:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..17    (D) OTHER INFORMATION: /product= "BB9526 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:138:    GTAGTCAGAAATGAAAT17    (2) INFORMATION FOR SEQ ID NO:139:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..18    (D) OTHER INFORMATION: /product= "BB9528 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:139:    GAGAAGAAGCTTCAAAGT18    (2) INFORMATION FOR SEQ ID NO:140:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..20    (D) OTHER INFORMATION: /product= "BB9535 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:140:    CTTAGTCAAGGCGATGACAC20    (2) INFORMATION FOR SEQ ID NO:141:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..20    (D) OTHER INFORMATION: /product= "BB9538 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:141:    GTCAGCACAGGCTTGTCTCG20    (2) INFORMATION FOR SEQ ID NO:142:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..17    (D) OTHER INFORMATION: /product= "BB9539 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:142:    TGGGTCAGAACAGACTT17    (2) INFORMATION FOR SEQ ID NO:143:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB9540 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:143:    CATTCTTCAGCTGGGTCAG19    (2) INFORMATION FOR SEQ ID NO:144:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..20    (D) OTHER INFORMATION: /product= "BB9541 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:144:    ATTTTTGAACAGCTTCTTCA20    (2) INFORMATION FOR SEQ ID NO:145:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..22    (D) OTHER INFORMATION: /product= "BB9542 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:145:    CATATTTTTGAGCCCATTCTTC22    (2) INFORMATION FOR SEQ ID NO:146:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..21    (D) OTHER INFORMATION: /product= "BB10374 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:146:    TTTTTGAACCAATTCTTCAGA21    (2) INFORMATION FOR SEQ ID NO:147:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..21    (D) OTHER INFORMATION: /product= "BB10375 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:147:    CAGAAACATAATCTTGAACCC21    (2) INFORMATION FOR SEQ ID NO:148:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB10376 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:148:    GTCAGAAACATCTTTTTGA19    (2) INFORMATION FOR SEQ ID NO:149:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB10377 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:149:    GTGTAAGAATCACAACAAG19    (2) INFORMATION FOR SEQ ID NO:150:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..23    (D) OTHER INFORMATION: /product= "BB11235 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:150:    GAAACAACAAGCTTCTGGAGTGT23    (2) INFORMATION FOR SEQ ID NO:151:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB10379 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:151:    ATTAGGCTTCCAATTCCAA19    (2) INFORMATION FOR SEQ ID NO:152:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..22    (D) OTHER INFORMATION: /product= "BB10380 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:152:    ATTAGGCAGAATCTTCCAAGTC22    (2) INFORMATION FOR SEQ ID NO:153:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..20    (D) OTHER INFORMATION: /product= "BB10381 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:153:    CAATTCCAATCTAGAAACAT20    (2) INFORMATION FOR SEQ ID NO:154:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB10382 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:154:    CATTGAGATTCAGTTTCAA19    (2) INFORMATION FOR SEQ ID NO:155:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB10383 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:155:    GTCAAGAAGTTGACACCTG19    (2) INFORMATION FOR SEQ ID NO:156:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..21    (D) OTHER INFORMATION: /product= "BB10964 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:156:    GCGCTTAGTGTTGAAGATGAC21    (2) INFORMATION FOR SEQ ID NO:157:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..27    (D) OTHER INFORMATION: /product= "BB10385 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:157:    GTAAGAGAAACATTGACAAGCGGTTGG27    (2) INFORMATION FOR SEQ ID NO:158:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..24    (D) OTHER INFORMATION: /product= "BB10386 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:158:    TTGAACCCATTGTTGAGATGGGTC24    (2) INFORMATION FOR SEQ ID NO:159:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..21    (D) OTHER INFORMATION: /product= "BB10529 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:159:    GTTTCAAAGTATTGAGCAATG21    (2) INFORMATION FOR SEQ ID NO:160:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..26    (D) OTHER INFORMATION: /product= "BB10530 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:160:    GATGACACCTGGTTCGGAACATTGAG26    (2) INFORMATION FOR SEQ ID NO:161:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..26    (D) OTHER INFORMATION: /product= "BB10531 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:161:    CTTGTCTCGAGCGTTCAGTCAAGAAG26    (2) INFORMATION FOR SEQ ID NO:162:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..25    (D) OTHER INFORMATION: /product= "BB10532 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:162:    GACTTGTCTCGATTCCTTAGTCAAG25    (2) INFORMATION FOR SEQ ID NO:163:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..27    (D) OTHER INFORMATION: /product= "BB10533 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:163:    CCATTCTTCAGATGGTGGAGCACAGAC27    (2) INFORMATION FOR SEQ ID NO:164:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..19    (D) OTHER INFORMATION: /product= "BB10534 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:164:    GCAGACAATTGCAAGTCAG19    (2) INFORMATION FOR SEQ ID NO:165:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..21    (D) OTHER INFORMATION: /product= "BB10535 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:165:    GTAGTCAGCCAAGAAATTTTG21    (2) INFORMATION FOR SEQ ID NO:166:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..21    (D) OTHER INFORMATION: /product= "BB10536 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:166:    GTAGTCAGCGACGAAATTTTG21    (2) INFORMATION FOR SEQ ID NO:167:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..22    (D) OTHER INFORMATION: /product= "BB10195 oligomer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:167:    GGAATTTGTTCAGAGGTGTAAG22    (2) INFORMATION FOR SEQ ID NO:168:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..17    (D) OTHER INFORMATION: /product= "BB5769 primer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:168:    GCATTCTGACATCCTCT17    (2) INFORMATION FOR SEQ ID NO:169:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..31    (D) OTHER INFORMATION: /product= "BB6040 primer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:169:    CGTTAAAATCAACAACTTGTCAATTGGAACC31    (2) INFORMATION FOR SEQ ID NO:170:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..17    (D) OTHER INFORMATION: /product= "BB6296 primer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:170:    GGAAATCTCACAGATCT17    (2) INFORMATION FOR SEQ ID NO:171:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..24    (D) OTHER INFORMATION: /product= "BB8461 primer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:171:    GAAGGAAATCTCATCGTTTGAATA24    (2) INFORMATION FOR SEQ ID NO:172:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..17    (D) OTHER INFORMATION: /product= "BB8740 primer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:172:    GCTAATGCGGAGGATGC17    (2) INFORMATION FOR SEQ ID NO:173:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..31    (D) OTHER INFORMATION: /product= "BB6394 primer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:173:    CCGGCATTACAACTTATCGATAAGCTTGCAC31    (2) INFORMATION FOR SEQ ID NO:174:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..17    (D) OTHER INFORMATION: /product= "BB6037 primer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:174:    GCGCATTGTTAGATTTC17    (2) INFORMATION FOR SEQ ID NO:175:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..24    (D) OTHER INFORMATION: /product= "BB6841 primer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:175:    CTTATCGATCAACTTGCACAAACG24    (2) INFORMATION FOR SEQ ID NO:176:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..28    (D) OTHER INFORMATION: /product= "BB6189 primer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:176:    GTCATGTCTAAGGCGGATCCTTATTAAC28    (2) INFORMATION FOR SEQ ID NO:177:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..24    (D) OTHER INFORMATION: /product= "BB8661 primer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:177:    GAGAATGGCAACAACTTATGCATT24    (2) INFORMATION FOR SEQ ID NO:178:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..17    (D) OTHER INFORMATION: /product= "BB6038 primer"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:178:    CCAACATCAATACAACC17    __________________________________________________________________________

We claim:
 1. A method of inhibiting proliferation of stem cells in apatient capable of benefitting from such inhibition, comprisingadminstering to said patient a therapeutically effective amount of acomposition comprising an LD78 or MIP-1α analogue of a wild-type LD78 orMIP-1α molecule, the analogue having stem cell inhibition (SCI) activityand being substantially incapable at physiological ionic strength offorming a stable multimer higher than a dodecamer as determined bySedimentation Equilibrium Analytical Ultracentrifugation (AUC) and apharmaceutically acceptable carrier.
 2. The method of claim 1, whereinsaid analogue, which, relative to wild-type LD78 or MIP-1α, one or moreamino acid residues involved in promoting and/or stabilizing associationof the components of a dimeric, tetrameric, dodecameric or higher ordercomplex is altered to have a lesser promoting and/or stabilizing effect.3. The method of claim 2, wherein said alteration is a substitution. 4.The method of claim 1, wherein said analogue is substantially incapableat physiological ionic strength of forming a stable multimer higher thana tetramer.
 5. The method of claim 4, wherein said analogue atphysiological ionic strength forms a substantially homogeneouspopulation of tetramers.
 6. The method of claim 1, wherein said analogueis substantially incapable at physiological ionic strength of forming astable multimer higher than a dimer.
 7. The method of claim 6, whereinsaid analogue is substantially incapable at physiological ionic strengthof forming a stable multimer higher than a monomer.
 8. A method ofinhibiting proliferation of stem cells in a patient, comprisingadministering to said patient a therapeutic effective amount of aproteinaceous molecule with stem cell inhibition (SCI) activity, themolecule being an analogue of a chemotactic cytokine superfamilymolecule having SCI activity and a tendency to aggregate atphysiological ionic strength, the analogue being substantially incapableat physiological ionic strength of forming a stable multimer higher thana dodecamer as determined by Sedimentation Equilibrium AnalyticalUltracentrifugation (AUC), wherein the analogue is an LD78 analogue witha mutation at one or more of the amino acid residues with respect towild-type LD78 selected from the group consisting of: Ser1, Leu2, Ala3,Ala4, Asp5, Thr6, Ala9, Phe12, Ser13, Tyr14, Ser16, Arg17, Gln18, Ile19,Pro20, Gln21, Phe23, Ile24, Asp26, Tyr 27, Phe28, Glu29, Ser31, Ser32,Gln33, Ser35, Lys36, Pro37, Gly38, Val39, Ile40, Leu42, Thr43, Lys44,Arg45, Ser46, Arg47, Gln48, Asp52, Glu55, Glu56, Gln59, Lys60, Tyr61,Val62, Asp64, Leu65, Glu66, Leu67, Ser68 and Ala69, and apharmaceutically acceptable carrier.
 9. The method of claim 8, whereinthe mutation is a substitution and wherein there are only two mutationsubstitutions in said molecule.
 10. A method of inhibiting proliferationof stem cells in a patient, comprising administering to said patient atherapeutic effective amount of a proteinaceous molecule with stem cellinhibition (SCI) activity, the molecule being an analogue of achemotactic cytokine superfamily molecule having SCI activity and atendency to aggregate at physiological ionic strength, the analoguebeing substantially incapable at physiological ionic strength of forminga stable multimer higher than a dodecamer as determined by SedimentationEquilibrium Analytical Ultracentrifugation (AUC), wherein the analogueis an LD78 analogue and has at least one of the following substitutionswith respect to wild-type LD78, wherein the substitutions are selectedfrom the group consisting of: (a) Ile24>Asn, (b) Tyr27>Asn, (c)Phe28>Glu, (d) Glu29>Arg, (e) Lys44>Glu and (f) Arg45>Glu, and apharmaceutically acceptable carrier.
 11. The method of claim 10, whereinthe analogue possesses both Lys44>Glu and Arg45>Gln substitutions.
 12. Amethod of inhibiting proliferation of stem cells in a patient,comprising administering to said patient a therapeutic effective amountof a proteinaceous molecule with stem cell inhibition (SCI) activity,the molecule being an analogue of a chemotactic cytokine superfamilymolecule having SCI activity and a tendency to aggregate atphysiological ionic strength, the analogue being substantially incapableat physiological ionic strength of forming a stable multimer higher thana dodecamer as determined by Sedimentation Equilibrium AnalyticalUltracentrifugation (AUC), wherein the analogue is an LD78 analogue andhas at least one of the following substitutions with respect towild-type LD78, wherein the substitutions are selected from the groupconsisting of: (a) Lys44>Glu with Arg45>Gln, (b) Arg47>Glu, (c)Phe28>Glu, (d) Phe28>Glu with Gln48>Glu, (e) Phe28>Glu with Arg47>Glu,(f) Arg17>Ser with Gln18>Glu, (g) Phe12>Ala, (h) Val39>Ala, (i)Ile40>Ala, (j) Asp26>Ala with Glu29>Arg and Arg47>Glu, (k) Arg17>Ser,(l) Glu29>Arg, (m) Gln18>Glu, (n) Asp26>Ser, (o) Gln48>Ser, (p)Thr15>Ala, (q) Gln21>Ser, (r) Phe23>Ala, (s) Ser32>Ala, (t) Ala51>Ser,(u) Ala4>Glu, (v) Phe12>Asp, (w) Asp26>Gln, (x) Lys36>Glu, (y)Lys44>Glu, (z) Arg45>Glu, (aa) Glu66>Gln, (bb) Phe12>Gln, (cc)Lys44>Ser, (dd) Arg17>Glu with Gln18>Glu, (ee) Asp26>Ala, (ff)Glu66>Ser, and, (gg) Ile19>Ala, and a pharmaceutically acceptablecarrier.
 13. The method of claim 12, wherein said molecule is an LD78analogue having the following substitution with respect to wild-typeLD78: Glu66>Ser.
 14. The method of claim 12, wherein said molecule is anLD78 analogue having both the following substitution with respect towild-type LD78: Ile19>Ala and Val39>Ala.
 15. A method of inhibitingproliferation of stem cells in a patient undergoing chemotherapy and/orradiotherapy or in a patient suffering from hyperproliferative stem celldisorders, comprising administering to said patient a therapeuticeffective amount of a proteinaceous molecule with stem cell inhibition(SCI) activity, the molecule being an analogue of a chemotactic cytokinesuperfamily molecule having SCI activity and a tendency to aggregate atphysiological ionic strength, the analogue being substantially incapableat physiological ionic strength of forming a stable multimer higher thana dodecamer as determined by Sedimentation Equilibrium AnalyticalUltracentrifugation (AUC), wherein the analogue is an LD78 analogue witha substitution mutation of aspartic acid to alanine at amino acidresidues 26 with respect to wild-type LD78, and a pharmaceuticallyacceptable carrier.
 16. A method of inhibiting proliferation of stemcells in a patient, comprising administering to said patient atherapeutic effective amount of a proteinaceous molecule with stem cellinhibition (SCI) activity, the molecule being an analogue of achemotactic cytokine superfamily molecule having SCI activity and atendency to aggregate at physiological ionic strength, the analoguebeing substantially incapable at physiological ionic strength of forminga stable multimer higher than a dodecamer as determined by SedimentationEquilibrium Analytical Ultracentrifugation (AUC), wherein the analogueis an MIP-1α analogue with a mutation at one or more of the amino acidresidues with respect to wild-type MIP-1 α selected from the groupconsisting of: Ala1, Pro2, Tyr3, Gly4, Ala5, Asp6, Thr7, Ala10, Phe13,Ser14, Tyr15, Ser16, Arg17, Lys 18, Ile19, Pro20, Arg21, Phe23, Ile24,Asp26, Phe28, Glu29, Ser3, Ser32, Leu33, Ser35, Gln36, Pro37, Gly38,Val39, Ile40, Leu42, Thr43, Lys44, Arg45, Asn46, Arg47, Gln48, Asp52,Glu55, Thr56, Gln59, Glu60, Tyr61, Ile62, Asp64, Leu65, Glu66, Leu67,Asn68 and Ala69, and a pharmaceutically acceptable carrier.
 17. Themethod of claim 16, wherein the mutation is a substitution and whereinthere are only two mutation substitutions in said molecule.